Peptide pharmaceuticals

ABSTRACT

Described herein are methods of syntheses and therapeutic uses of covalently modified peptides and/or proteins. The covalently modified peptides and/or proteins allow for improved pharmaceutical properties of peptide and protein-based therapeutics.

INCORPORATION BY REFERENCE

This application is a continuation of U.S. application Ser. No.14/646,246, which was filed May 20, 2015, pursuant to 35 U.S.C. § 371 asa United States National Phase Application of International ApplicationSerial No. PCT/US2013/071067, which claims the benefit of U.S.Provisional Patent Application Ser. No. 61/728,667, filed Nov. 20, 2012,each of which are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 30, 2015, isnamed 38617-701-302_SL and is 695,244 bytes in size.

FIELD OF THE INVENTION

Covalently modified peptide and protein analogs allow for improvedpharmaceutical properties of peptide and/or protein-based therapeutics.

SUMMARY OF THE INVENTION

Peptide and/or protein pharmaceuticals suffer from several limitationsin their use in medicine (Nestor, J. J., Jr. (2007) ComprehensiveMedicinal Chemistry II 2: 573-601)—short duration of action, poorbioavailability, and lack of receptor subtype selectivity. In addition,peptides and/or proteins are unstable in formulations, being subject toaggregation. In some instances, aggregation of peptides and/or proteinsleads to the development of an immunological response to both native andforeign peptides or proteins.

Described herein is a method and reagents for covalently modifyingpeptides and/or proteins to generate products with improvedpharmaceutical properties. In some instances, covalently modifiedpeptides and/or proteins allow for improved stability, bioavailability,selectivity, and duration of effect in peptide and/or protein-basedtherapeutics.

Described herein are certain covalently modified peptides and/orproteins with improved pharmaceutical properties. In some instances,these covalently modified peptides and/or proteins allow for improvedstability, bioavailability, selectivity, and duration of effect inpeptide and/or protein-based therapeutics.

In some embodiments, the covalently modified peptides and/or proteinsdescribed herein are attached to glycoside surfactants. In one aspect,the covalently modified peptides and/or proteins are attached to alkylglycosides. In one aspect, the covalently modified peptides and/orproteins are attached to an alkyl glycoside surfactant wherein thepeptide and/or protein is attached to the glycoside in the surfactantand the glycoside is then attached to a hydrophobic and/or alkyl group.Provided herein, in some embodiments, are reagents and intermediates forthe covalent modification of peptides and/or proteins through theincorporation of surfactants such as alkyl glycosides.

Provided herein are peptide products comprising a surfactant X,covalently attached to a peptide, the peptide comprising a linker aminoacid U and at least one other amino acid:

wherein X is

-   -   wherein    -   A is a hydrophobic group; and    -   B is a hydrophilic group covalently attached to the peptide via        a linker amino acid U.

In some embodiments, the peptide product is synthesized by reaction of afunctionalized surfactant with the peptide as described herein. In someembodiments, the peptide product is synthesized by reaction of afunctionalized surfactant with a reversibly-protected linker amino acidas described herein followed by reaction with one or more amino acids toform a surfactant-modified peptide product. In some embodiments, U is aterminal amino acid of the peptide. In some embodiments, U is anon-terminal amino acid of the peptide.

In some embodiments, A is a substituted or unsubstituted C₁-C₃₀ alkylchain, a substituted or unsubstituted alkoxyaryl group, a substituted orunsubstituted aralkyl group or a steroid nucleus containing moiety. Insome embodiments, A is a substituted or unsubstituted C₈-C₂₀ alkylchain, a substituted or unsubstituted 1-alkoxyaryl group, a substitutedor unsubstituted 1-aralkyl group, or a steroid nucleus containingmoiety. In some embodiments, A is a substituted or unsubstituted C₁₀-C₂₀alkyl chain, a substituted or unsubstituted 1-alkoxyaryl group, asubstituted or unsubstituted 1-aralkyl group, or a steroid nucleuscontaining moiety.

In some embodiments, B is a polyol. In some embodiments, the polyol is asaccharide. In some embodiments, the saccharide is a monosaccharide, adisaccharide, or a polysaccharide. In some embodiments, the saccharideis selected from glucose, mannose, maltose, a glucuronic acid,galacturonic acid, diglucuronic acid and maltouronic acid.

In some embodiments, the surfactant is a 1-alkyl glycoside classsurfactant.

In some embodiments, the hydrophilic group in the surfactant is attachedto the peptide via an amide bond.

In some embodiments of the peptide product, the surfactant is comprisedof 1-eicosyl beta-D-glucuronic acid, 1-octadecyl beta-D-glucuronic acid,1-hexadecyl beta-D-glucuronic acid, 1-tetradecylbeta D-glucuronic acid,1-dodecyl beta D-glucuronic acid, 1-decyl beta-D-glucuronic acid,1-octyl beta-D-glucuronic acid, 1-eicosyl beta-D-diglucuronic acid,1-octadecyl beta-D-diglucuronic acid, 1-hexadecyl beta-D-diglucuronicacid, 1-tetradecyl beta-D-diglucuronic acid, 1-dodecylbeta-D-diglucuronic acid, 1-decyl beta-D-diglucuronic acid, 1-octylbeta-D-diglucuronic acid, or functionalized 1-ecosyl beta-D-glucose,1-octadecyl beta-D-glucose, 1-hexadecyl beta-D-glucose, 1-tetradecylbeta-D-glucose, 1-dodecyl beta-D-glucose, 1-decyl beta-D-glucose,1-octyl beta-D-glucose, 1-eicosyl beta-D-maltoside, 1-octadecylbeta-D-maltoside, 1-hexadecyl beta-D-maltoside, 1-dodecylbeta-D-maltoside, 1-decyl beta-D-maltoside, 1-octyl beta-D-maltoside,and the like, and the peptide product is prepared by formation of alinkage between the aforementioned groups and a group on the peptide(e.g., a —COOH group in the aforementioned groups and an amino group ofthe peptide).

In some embodiments, a combination of a hydrophilic with a hydrophobicgroup generates a surfactant. In some embodiments, the surfactant is anionic surfactant. In some embodiments, the surfactant is a non-ionicsurfactant. In some embodiments, the hydrophobic group is a substitutedor unsubstituted C₁-C₃₀ alkyl chain or an aralkyl chain. In someembodiments the hydrophobic group is a chain having mixed hydrophobicand hydrophilic properties, for example a polyethylene glycol (PEG)group.

In some embodiments, the linker amino acid is a natural D- or L-aminoacid. In some embodiments, the linker amino acid is an unnatural aminoacid. In some embodiments, the linker amino acid is selected from Lys,Cys, Orn, Asp, Glu or an unnatural amino acid, comprising a functionalgroup used for covalent attachment to the surfactant X. In someembodiments, the linker amino acid is selected from Lys, Cys, Orn, or anunnatural amino acid, comprising a functional group used for covalentattachment to the surfactant X. In some embodiments, the functionalgroup used for covalent attachment to a surfactant is —NH₂, —SH, —OH,—N₃, haloacetyl, a —(CH2)_(m)-maleimide or an acetylenic group, whereinm is 1-10.

In some embodiments, the peptide is an opioid peptide. In someembodiments, the peptide and/or protein product contains a covalentlylinked alkyl glycoside. In some of such embodiments, the peptide and/orprotein product contains a covalently linked alkyl glycoside that is a1-O-alkyl glucuronic acid of alpha or beta configuration. In some ofsuch embodiments, the peptide and/or protein product comprises acovalently linked alkyl glycoside that is a 1-O-alkyl glucuronic acidand the alkyl chain is a C₁ to C₂₀ alkyl chain.

In some embodiments, the peptide product has a structure of Formula IA:

aa₁-aa₂-aa₃-aa₄-aa₅-Z  Formula IA

-   -   wherein:        -   each of aa₁, aa₂, aa₃, aa₄, and aa₅ is independently absent,            a D- or L-natural or unnatural amino acid, an N-alkylated            amino acid, an N-acetylated amino acid, a CαR³ amino acid, a            Ψ-amino acid, or a linker amino acid U covalently attached            to the surfactant X;        -   Z is —OH, —NH₂ or —NHR³;        -   each R³ is independently substituted or unsubstituted C₁-C₁₂            branched or straight chain alkyl, a PEG chain of less than            10 Da, or substituted or unsubstituted aralkyl chain;        -   provided that one, or at least one of aa₁, aa₂, aa₃, aa₄,            and aa₅ is the linker amino acid U covalently attached to            the surfactant X;        -   and further provided that not all of aa₁, aa₂, aa₃, aa₄, and            aa₅ are absent.

In one aspect, provided herein is a peptide product having a structureof Formula II:

aa₁-aa₂-aa₃-aa₄-aa₅-Z  Formula II

-   -   wherein:        -   each of aa₁, aa₂, aa₃, aa₄, and aa₅ is independently absent,            a D- or L-natural or unnatural amino acid, an N-alkylated            amino acid, an N-acetylated amino acid, a CαR³ amino acid, a            Ψ-amino acid, or a linker amino acid U covalently attached            to a surfactant X;        -   Z is —OH, —NH₂ or —NHR³;        -   each R³ is independently substituted or unsubstituted C₁-C₁₂            branched or straight chain alkyl, a PEG chain of less than            10 Da, or substituted or unsubstituted aralkyl chain; and        -   X is

-   -   -   -   wherein                -   A is a hydrophobic group; and                -   B is a hydrophilic group covalently attached to the                    peptide via a linker amino acid U;

        -   provided that one, or at least one of aa₁, aa₂, aa₃, aa₄,            and aa₅ is the linker amino acid U covalently attached to            the surfactant X;

        -   and further provided that not all of aa₁, aa₂, aa₃, aa₄, and            aa₅ are absent.

In one aspect, provided herein is a peptide product that has a structureof Formula III:

Formula III (SEQ. ID. NO. 1) aa₁-aa₂-aa₃-aa₄-aa₅-Z

-   -   wherein:        -   aa₁ is Tyr, Dmt, N—R³-Tyr, N—R³-Dmt, N—(R³)₂-Tyr, or            N—(R³)₂-Dmt;        -   aa₂ is Pro, D-Arg, D-Cit, D-U(X), D-Ala, Tic, or            Tic(Ψ[CH2-NH]);        -   aa₃ is Phe, Trp, Tmp, D- or L-Nal(1), D- or L-Nal(2),            CaMePhe, or Ψ-Phe;        -   aa₄ is Phe, Tmp, D- or L-Nal(1), D- or L-Nal(2), U(X), D- or            L-CαMeU(X);        -   aa₅ is absent or Pro, Aib, U(X), D- or L-CαMeU(X); and        -   U is a dibasic natural or unnatural amino acid, a natural or            unnatural amino acid comprising a thiol, an unnatural amino            acid comprising a —N₃ group, an unnatural amino acid            comprising an acetylenic group, or an unnatural amino acid            comprising a —NH—C(═O)—CH₂—Br or a —(CH₂)_(m)-maleimide,            wherein m is 1-10, used for covalent attachment to the            surfactant X.

In some embodiments of peptide products of Formula I, Formula II orFormula III described above and herein, X has the structure:

-   -   wherein:        -   A is a substituted or unsubstituted C₁-C₃₀ alkyl chain, a            substituted or unsubstituted alkoxyaryl group, a substituted            or unsubstituted aralkyl group, or a steroid nucleus            containing moiety;        -   R^(1b), R^(1c), and R^(1d) are each, independently at each            occurrence, H, a protecting group, a saccharide, a            substituted or unsubstituted C₁-C₃₀ alkyl group, a            substituted or unsubstituted alkoxyaryl group, or a            substituted or unsubstituted aralkyl group;        -   W¹ is independently, at each occurrence, —CH₂—, —CH₂—O—,            —(C═O), —(C═O)—O—, —(C═O)—NH—, —(C═S)—, —(C═S)—NH—, or            —CH₂—S—;        -   W² is —O—, or —S—;        -   R² is a bond, C₂-C₄-alkene, C₂-C₄-alkyne, or            —(CH₂)_(m)-maleimide, and        -   m is 1-10.

In some embodiments of peptide products of Formula I, Formula II orFormula III described above and herein, X has the structure:

Accordingly, in the embodiment described above, R² is a bond.

In some embodiments of peptide products of Formula I, Formula II orFormula III described above and herein, X has the structure:

In some embodiments of peptide products of Formula I, Formula II orFormula III described above and herein, X has the structure:

-   -   wherein:        -   A is a substituted or unsubstituted C₁-C₃₀ alkyl group, or a            steroid nucleus containing moiety;        -   R^(1b), R^(1c), and R^(1d) are each, independently at each            occurrence, H, a protecting group, a saccharide or a            substituted or unsubstituted C₁-C₃₀ alkyl group;        -   W¹ is —(C═O)—NH—;        -   W² is —O—;        -   R² is a bond.

In some embodiments of peptide products of Formula I, Formula II orFormula III described above and herein, X has the structure:

-   -   wherein:        -   A is a substituted or unsubstituted C₁-C₃₀ alkyl group;        -   R^(1b), R^(1c), and R^(1d) are H;        -   W¹ is —(C═O)—NH—;        -   W² is —O—; and        -   R² is a bond.

In some embodiments of peptide products of Formula I, Formula II orFormula III described above and herein, X is as described above and A isa substituted or unsubstituted C₁-C₂₀ alkyl group.

In some embodiments described above and herein, R^(1a) is a saccharide.In some embodiments, the saccharide is a galactose. In certainembodiments, the saccharide is an alpha-linked galactose. In otherembodiments, the saccharide is alpha-linked galactopyranose, beta-linkedgalactopyranose, alpha-linked galactofuranose, or beta-linkedgalactofuranose.

Also contemplated herein are alternate embodiments wherein X in FormulaI has the structure:

For instance, in an exemplary embodiment of the structure of X describedabove, W¹ is —S—, R² is a C₁-C₃₀ alkyl group, W² is S, R^(1a) is a bondbetween W² and a suitable moiety of an amino acid residue U within thepeptide (e.g., a thiol group in a cysteine residue of the peptide formsa thioether with X).

In another exemplary alternate embodiment of the structure of Xdescribed above, W¹ is —O—, R² is a C₁-C₃₀ alkyl group, W² is O, R^(1a)is a bond between W² and a suitable moiety of an amino acid residue Uwithin the peptide (e.g., a hydroxyl group in a serine or threonineresidue of the peptide forms an ether with X).

In some embodiments, a peptide product of Formula III is a product (SEQ.ID. NO. 149)

-   -   wherein:        -   U is a dibasic natural or unnatural amino acid;        -   X is a surfactant of the 1-alkyl glycoside class wherein            1-alkyl is substituted or unsubstituted C₁-C₂₀ alkyl or a            substituted or unsubstituted alkoxyaryl substituent;        -   Z is NH₂;        -   aa₁ is Tyr, Dmt, Nα-Me-Tyr, Nα-Me-Tyr, N,Nα-diMe-Tyr, or            N,Nα-diMe-Dmt;        -   aa₂ is Pro, D-Arg, D-Cit, D-U(X), D-Ala, Tic, or            Tic(Ψ[CH2-NH]);        -   aa₃ is Phe, Trp, Tmp, D- or L-Nal(1), D- or L-Nal(2),            CaMePhe, or Ψ-Phe;        -   aa₄ is Phe, Tmp, D- or L-Nal(1), D- or L-Nal(2), U(X), D- or            L-CαMeU(X);        -   aa₅ is absent or Pro, Aib, U(X), D- or L-CαMeU(X).

In some embodiments, a peptide product of Formula III is a product (SEQ.ID. NO. 150)

-   -   wherein:        -   X is comprised of 1-alkyl glucuronic acid or 1-alkyl            diglucuronic acid;        -   Z is NH₂;        -   aa₁ is Dmt;        -   aa₂ is Pro, D-Lys(X), Tic, or Tic(Ψ[CH2-NH]);        -   aa₃ is Phe, Tmp, D- or L-Nal(1), D- or L-Nal(2), or Ψ-Phe;        -   aa₄ is Phe, D- or L-Nal(1), D- or L-Nal(2), or Lys(X);        -   aa₅ is absent or Pro, Aib, Lys(X), D- or L-CαMeLys(X).

In some embodiments, a peptide product of Formula III is a product (SEQ.ID. NO. 151)

-   -   wherein:        -   aa₁ is Dmt;        -   aa₂ is Pro;        -   aa₃ is Phe, or Tmp;        -   aa₄ is Phe, or Lys(X);        -   aa₅ is absent or Aib, Lys(X), D- or L-CαMeLys(X).

In some embodiments, a peptide product of Formula III is a product (SEQ.ID. NO. 152)

-   -   wherein:        -   aa₁ is Dmt;        -   aa₂ is Pro;        -   aa₃ is Phe, or Tmp;        -   aa₄ is Phe, or Lys(X);        -   aa₅ is absent or Lys(X).

In some embodiments, a peptide product of Formula III is a product (SEQ.ID. NO. 153)

-   -   wherein:        -   U is a dibasic natural or unnatural amino acid;        -   X is a surfactant of the 1-alkyl glycoside class wherein the            1-alkyl group of the 1-alkyl glycoside is substituted or            unsubstituted C₁₋₂₀ alkyl or a substituted or unsubstituted            alkoxyaryl substituent;        -   aa₁ is Tyr, Dmt, N—R³-Tyr, N—R³-Tyr, N—(R³)₂-Tyr, or            N—(R³)₂-Dmt;        -   aa₂ is D-Arg, D-Cit, or D-U(X);        -   aa₃ is Phe, Trp, D- or L-Nal(1), D- or L-Nal(2), or Tmp;        -   aa₄ is Phe, Tmp, D- or L-Nal(1), D- or L-Nal(2), U(X), D- or            L-CαMeU(X);        -   aa₅ is absent, Pro, or U(X).

In some embodiments, a peptide product of Formula III is a product (SEQ.ID. NO. 154)

-   -   wherein:        -   X is a surfactant of the 1-alkyl glucuronic acid or 1-alkyl            diglucuronic acid class;        -   aa₁ is Dmt, Nα-Me-Dmt, or N,Nα-Me-Dmt;        -   aa₂ is D-Arg, D-Cit, D-Lys(X), or D-Orn(X);        -   aa₃ is Phe, or Tmp;        -   aa₄ is Phe, Tmp, Lys(X), or Orn(X);        -   aa₅ is absent or Pro, Lys(X), or Orn(X).

In some embodiments, a peptide product of Formula III is a product (SEQ.ID. NO. 155)

-   -   wherein:        -   U is a dibasic natural or unnatural amino acid;        -   X is a surfactant of the 1-alkyl glycoside class wherein            1-alkyl is substituted or unsubstituted C₁-C₂₀ alkyl or a            substituted or unsubstituted alkoxyaryloxy substituent;        -   Z is NH₂;        -   aa₁ is Tyr, Dmt, N—R³-Tyr, N—R³-Dmt, N—(R³)₂-Tyr, or            N—(R³)₂-Dmt;        -   aa₂ is Tic, or Tic(Ψ[CH2-NH]);        -   aa₃ is Ψ-Phe when aa2 is Tic(Ψ[CH2-NH]);        -   aa₄ is Phe, Tmp, D- or L-Nal(1), D- or L-Nal(2), or U(X);        -   aa₅ is absent, Pro, Aib, or U(X).

In some embodiments, a peptide product of Formula III is a product (SEQ.ID. NO. 156)

-   -   wherein:        -   X is a surfactant of the 1-alkyl glucuronic acid or 1-alkyl            diglucuronic acid class;        -   aa₁ is Tyr, Dmt, Nα-Me-Tyr, Nα-alkyl-Dmt, N,Nα-diMe-Tyr, or            N,Nα-Me-Dmt;        -   aa₂ is Tic, or Tic(Ψ[CH2-NH]);        -   aa₃ is Ψ-Phe when aa₂ is Tic(Ψ[CH2-NH]), Phe, or TMP;        -   aa₄ is Phe, Tmp, D- or L-Nal(1), D- or L-Nal(2), Lys(X), or            Orn(X);        -   aa₅ is absent or Aib, Lys(X), or Orn(X).

In some embodiments, a peptide product of Formula III is a product (SEQ.ID. NO. 157)

-   -   wherein:        -   X is comprised of 1-alkyl glucuronic acid or 1-alkyl            diglucuronic acid;        -   aa₂ is Tic, or Tic(Ψ[CH2-NH]);        -   aa₃ is Phe or Ψ-Phe;        -   aa₄ is Lys(X);        -   aa₅ is absent.

In some embodiments, a peptide product of Formula III is a product (SEQ.ID. NO. 158)

-   -   wherein:        -   X is comprised of 1-alkyl glucuronic acid;        -   aa₂ is Tic:        -   aa₃ is Phe;        -   aa₄ is Lys(X);        -   aa₅ is absent.

In some embodiments, a peptide product of Formula III is a product (SEQ.ID. NO. 159)

-   -   wherein:        -   X is comprised of a 1-alkyl glucuronic acid selected from            1-methyl beta-D-glucuronic acid, 1-octyl beta-D-glucuronic            acid, 1-dodecyl beta-D-glucuronic acid, 1-tetradecyl            beta-D-glucuronic acid, 1-hexadecyl beta-D-glucuronic acid,            and 1-octadecyl beta-D-glucuronic acid;        -   aa₂ is Tic;        -   aa₃ is Phe;        -   aa₄ is Lys(X);        -   aa₅ is absent.

In some embodiments, a peptide product of Formula III is

-   -   H-Dmt-Tic-Phe-Lys(N-epsilon-1-methyl beta-D-glucuronyl)-NH₂.        (SEQ. ID. NO. 78)

In some embodiments, a peptide product of Formula III is

-   -   H-Dmt-Tic-Phe-Lys(Nepsilon-1-octyl beta-D-glucuronyl)-NH₂. (SEQ.        ID. NO. 80)

In some embodiments, a peptide product of Formula III is

-   -   H-Dmt-Tic-Phe-Lys(Nepsilon-1-dodecyl beta-D-glucuronyl)-NH₂.        (SEQ. ID. NO. 79)

In some embodiments, a peptide product of Formula III is

-   -   H-Dmt-Tic-Phe-Lys(Nepsilon-1-tetradecyl beta-D-glucuronyl)-NH₂.        (SEQ. ID. NO. 160)

In some embodiments, a peptide product of Formula III is

-   -   H-Dmt-Tic-Phe-Lys(Nepsilon-1-hexadecyl beta-D-glucuronyl)-NH₂.        (SEQ. ID. NO. 82)

In some embodiments, a peptide product of Formula III is

-   -   H-Dmt-Tic-Phe-Lys(N-epsilon-1-octadecyl beta-D-glucuronyl)-NH₂.        (SEQ. ID. NO. 83)

In some embodiments, the peptide product is biologically active.

In a specific embodiment, provided herein is a compound selected fromcompounds of Table 1 in FIG. 1.

Also provided herein is a pharmaceutical composition comprising atherapeutically effective amount of a peptide product of Formula I, IIor III, or pharmaceutically acceptable salt thereof, and at least onepharmaceutically acceptable carrier or excipient.

Provided herein is a method of treating pain comprising administrationof a therapeutically effective amount of a peptide product of Formula I,II or III or compounds of Table 1 in FIG. 1.

A method for improving the pharmaceutical and medicinal behavior of apeptide, thereby improving its duration of action, bioavailability orits stability in formulation, comprising covalent attachment of asurfactant X to the peptide, wherein X is as described herein.

In some embodiments, a peptide product comprising a covalently linkedalkyl glycoside described herein is an analog of Leu-enkephalin. In someof such embodiments, the peptide product contains a covalently linked1-O-alkyl β-D-glucuronic acid and the peptide is an analog ofLeu-enkephalin.

In some embodiments, a peptide product comprising a covalently linkedalkyl glycoside described herein is an analog of opioid peptide DPDPE(Akiyama K, et al. (1985) Proc Natl Acad Sci USA 82:2543-7). In some ofsuch embodiments, the peptide product contains a covalently linked1-O-alkyl glucuronic acid and the peptide is an analog of opioid peptideDPDPE.

In some embodiments, a peptide product comprising a covalently linkedalkyl glycoside described herein is an analog of the D-amino acidcontaining natural product peptides dermorphin (Melchiorri, P. andNegri, L. (1996) Gen Pharmacol 27: 1099-1107) or deltorphin (Erspamer,V., et al. (1989) Proc Natl Acad Sci USA 86:5188-92). In some of suchembodiments, the peptide product contains a covalently linked 1-O-alkylβ-D-glucuronic acid and the peptide is an analog of dermorphin ordeltorphin.

In some embodiments, a peptide product comprising a covalently linkedalkyl glycoside is an analog of endomorphin-1 or -2. In some of suchembodiments, the peptide product comprises a covalently linked 1-O-alkylβ-D-glucuronic acid and the peptide is an analog of endomorphin(Lazarus, L. H. and Okada, Y. (2012) Expert Opin Ther Patents 22: 1-14).

In some embodiments, a peptide product comprising a covalently linkedalkyl glycoside described herein is an analog of opioid peptidedynorphin (James, I. F., et al. (1982) Life Sci 31:1331-4). In some ofsuch embodiments, the peptide product contains a covalently linked1-O-alkyl β-D-glucuronic acid and the peptide is an analog of dynorphin.

In some embodiments side chain functional groups of two different aminoacid residues are linked to form a cyclic lactam. For example, in someembodiments, a Lys side chain forms a cyclic lactam with the side chainof Glu. In some embodiments such lactam structures are reversed and areformed from a Glu and a Lys. Such lactam linkages in some instances areknown to stabilize alpha helical structures in peptides (Condon, S. M.,et al. (2002) Bioorg Med Chem 10: 731-736).

In some embodiments side chain functional groups of two different aminoacid residues with —SH containing side chains are linked to form acyclic disulfide. For example, in some embodiments, two penicillamine ortwo Cys side chains may be linked to constrain the conformation of apeptide (Akiyama K, et al. (1985) Proc Natl Acad Sci USA 82:2543-7) inorder to yield greater duration of action or greater receptorselectivity.

In a specific embodiment, the peptide products of Formula I, Formula IIor Fomula III, described above and herein have the following structure:

wherein A is a C₁-C₂₀ alkyl chain as described in Table 1 of FIG. 1, R′is a peptide as described in Table 1 of FIG. 1, W² of Formula V is —O—,and W¹ of Formula V is —(C═O)NH— and is part of an amide linkage to thepeptide R′. In some of such embodiments, A is a C₆-C₂₀ alkyl chain. Insome of such embodiments, A is a C₁-C₁₀ alkyl chain. In some of suchembodiments, A is a C₁₂-C₂₀ alkyl chain. In some of such embodiments, Ais a C₁₂-C₁₈ alkyl chain.

In embodiments described above, an amino moiety of an amino acid and/ora peptide R′ (e.g., an amino group of an amino acid residue such as alysine, or a lysine within the peptide R′) is used to form a covalentlinkage with a compound of structure:

wherein A is a C₁-C₂₀ alkyl chain as described above and in Table 1 ofFIG. 1.

In such cases, the amino acid residue having an amino moiety (e.g., aLysine within the peptide R′) which is used to form a covalent linkageto the compound of Formula A described above, is a linker amino acid Uwhich is attached to a surfactant X. Accordingly, as one example,Lys(C12) of Table 1 of FIG. 1 has the following structure:

Also contemplated within the scope of the embodiments presented hereinare peptide products of Formula I derived from maltouronic acid-basedsurfactants prepared by covalent linkage of a peptide to either or bothcarboxylic acid groups. Thus, as one example, peptides in Table 1 ofFIG. 1 comprise a lysine linker amino acid bonded to a maltouronic acidbased surfactant X and having a structure:

Also contemplated within the scope of the embodiments presented hereinare peptide products of Formula I-A derived from melibiouronicacid-based surfactants through binding at the carboxylic acid function.Thus, as one example, peptides in Table 1 of FIG. 1 or Table 2 of FIG. 2or Table 3 of FIG. 3 comprise a lysine linker amino acid bonded to amelibiouronic acid based surfactant X and having a structure:

It will be understood that in one embodiment, compounds of Formula I areprepared by attaching a lysine to a group X, followed by attachment ofadditional amino acid residues and/or peptides are attached to thelysine-X compound to obtain compounds of Formula I. It will beunderstood that other natural or non-natural amino acids describedherein are also suitable for attachment to the surfactant X and aresuitable for attaching additional amino acid/peptides to obtaincompounds of Formula I. It will be understood that in anotherembodiment, compounds of Formula I are prepared by attaching a fulllength or partial length peptide to a group X, followed by optionalattachment of additional amino acid residues and/or peptides areattached to obtain compounds of Formula I.

Also provided herein is a pharmaceutical composition comprising atherapeutically effective amount of a peptide product described above,or acceptable salt thereof, and at least one pharmaceutically acceptablecarrier or excipient. In some embodiments, the carrier is anaqueous-based carrier. In some embodiments, the carrier is anonaqueous-based carrier. In some embodiments, the nonaqueous-basedcarrier is a hydrofluoroalkane-like solvent comprising sub-micronanhydrous α-lactose, or other excipients.

Contemplated within the scope of embodiments presented herein is thereaction of an amino acid and/or a peptide comprising a linker aminoacid U bearing a nucleophile, and a group X comprising a leaving groupor a functional group that can be activated to contain a leaving group,for example a carboxylic acid, or any other reacting group, therebyallowing for covalent linkage of the amino acid and/or peptide to asurfactant X via the linker amino acid U to provide a peptide product ofFormula I.

Also contemplated within the scope of embodiments presented herein isthe reaction of an amino acid and/or a peptide comprising a linker aminoacid U bearing a leaving group or a functional group that can beactivated to contain a leaving group, for example a carboxylic acid, orany other reacting group, and a group X comprising a nucleophilic group,thereby allowing for covalent linkage of the amino acid and/or peptideto a surfactant X via the linker amino acid U to provide a peptideproduct of Formula I.

It will be understood that, in one embodiment, Compounds of Formula Iare prepared by reaction of a linker amino acid U with X, followed byaddition of further residues to U to obtain the peptide product ofFormula I. It will be understood that in an alternative embodiment,Compounds of Formula I are prepared by reaction of a suitable peptidecomprising a linker amino acid U with X, followed by optional additionof further residues to U, to obtain the peptide product of Formula I.

Further provided herein are certain intermediates and/or reagents thatare suitable for synthesis of peptide products described herein. Incertain embodiments, such intermediates and/or reagents arefunctionalized surfactants that allow for covalent linkage with apeptide. In certain embodiments, such intermediates are functionalized1-alkyl glycoside surfactants that allow for covalent linkage with apeptide. In certain embodiments, such intermediates are functionalized1-alkyl glycoside surfactants linked to reversibly-protected linkeramino acids that allow for covalent linkage with other amino acids toform a peptide. It will be understood that a suitably functionalizedsurfactant is covalently coupled to a peptide via reaction with anappropriately matched functional group that is on a linker amino acid.

In some embodiments, intermediates suitable for synthesis of peptideproducts described herein are compounds of Formula IV. Accordingly,provided herein are intermediates and/or compounds of Formula IV:

-   -   wherein:    -   R^(1a) is independently at each occurrence a bond, H, a        protecting group, a natural or unnatural amino acid, a        substituted or unsubstituted C₁-C₃₀ alkyl hydrophobic group, a        substituted or unsubstituted alkoxyaryl group, a substituted or        unsubstituted aralkyl group, or a steroid nucleus containing        moiety;    -   R^(1b), R^(1c), and R^(1d) are each independently at each        occurrence a bond, H, a protecting group, a natural or unnatural        amino acid, a substituted or unsubstituted C₁-C₃₀ alkyl        hydrophobic group, a substituted or unsubstituted alkoxyaryl        group, or a substituted or unsubstituted aralkyl group;    -   W¹ is —CH₂—, —CH₂—O—, —(C═O), —(C═O)—O—, —(C═O)—NH—, —(C═S)—,        —(C═S)—NH—, or —CH₂—S—;    -   W² is —O—, —CH₂— or —S—;    -   R² is H, a protecting group, a natural or unnatural amino acid,        a substituted or unsubstituted C₁-C₃₀ alkyl hydrophobic group, a        substituted or unsubstituted alkoxyaryl group, a substituted or        unsubstituted aralkyl group, —NH₂, —SH, C₂-C₄-alkene,        C₂-C₄-alkyne, —NH(C═O)—CH₂—Br, —(CH₂)_(m)-maleimide, or —N₃;    -   n is 1, 2 or 3; and    -   m is 1-10.

In some embodiments, each natural or unnatural amino acid isindependently a reversibly protected or free linker amino acid. In someof such embodiments, the linker amino acid is a reversibly protected orfree lysine.

In some embodiments of Formula IV,

-   -   n is 1;    -   W¹ is —(C═O)—;    -   R^(1a) is a substituted or unsubstituted C₁-C₃₀ alkyl        hydrophobic group, a substituted or unsubstituted 1-alkoxyaryl        group, or a substituted or unsubstituted 1-aralkyl group,    -   R² is a reversibly-protected lysine of D- or L-configuration.

In some embodiments of Formula IV,

-   -   n is 1;    -   W¹ is —(C═O)—;    -   R^(1a) is a substituted or unsubstituted C₈-C₃₀ alkyl        hydrophobic group, a substituted or unsubstituted 1-alkoxyaryl        group, or a substituted or unsubstituted 1-aralkyl group,    -   R² is a reversibly protected lysine of D- or L-configuration.

In some of such embodiments, R^(1a) is an octyl, decyl, dodecyl,tetradecyl, or hexadecyl group.

In some embodiments of Formula IV,

-   -   n is 1;    -   W¹ is —(C═O)—NH— or —(C═O)—O—;    -   R² is a substituted or unsubstituted C₁-C₃₀ alkyl hydrophobic        group, a substituted or unsubstituted 1-alkoxyaryl group, or a        substituted or unsubstituted 1-aralkyl group,    -   R^(1a) is a reversibly protected serine or threonine of D- or        L-configuration.

In some of such embodiments, R² is an octyl, decyl, dodecyl, tetradecylor hexadecyl group.

In some embodiments of Formula IV,

-   -   n is 1;    -   m is 1-6;    -   W¹ is —CH₂—;    -   R^(1a) is a C₁-C₃₀ alkyl hydrophobic group, a 1-alkoxyaryl        group, or a 1-aralkyl group,    -   R² is —N₃, NH₂, —C₂-alkyne, —(CH₂)_(m)-maleimide,        NH—(C═O)—CH₂—Br, or    -   NH—(C═O)—CH₂—I.

In some embodiments of Formula IV,

-   -   n is 1;    -   W¹ is —(C═O)—O—;    -   R² is H,    -   R^(1a) is a substituted or unsubstituted C₁-C₃₀ alkyl        hydrophobic group.

In some embodiments of Formula IV, R² is attached to the peptide or isan amino acid residue in the peptide. In some of such embodiments, R² isa reversibly protected or free lysine.

In some embodiments of Formula IV, n is 1. In some embodiments ofFormula IV, n is 2, and a first glycoside is attached to a secondglycoside via a bond between W² of the first glycoside and any one ofOR^(1b), OR^(1c) or OR^(1d) of the second glycoside.

In some embodiments of Formula IV, n is 3, and a first glycoside isattached to a second glycoside via a bond between W² of the firstglycoside and any one of OR^(1b), OR^(1c) or OR^(1d) of the secondglycoside, and the second glycoside is attached to a third glycoside viaa bond between W² of the second glycoside and any one of OR^(1b),OR^(1c) or OR^(1d) of the third glycoside.

Provided herein is a method for synthesizing a peptide product describedabove, comprising sequential steps of

-   -   (a) coupling a compound of Formula IV to the peptide; and    -   (b) optionally deprotecting the coupled peptide of step (a).

In some embodiments, the deprotecting comprises the use of mild acid andor mild base treatments. In some embodiments of the methods, thedeprotecting comprises the use of strong acids.

In some embodiments, the method further comprises the steps ofchromatography, desalting of intermediates by reversed-phase,high-performance liquid chromatography or ion exchange chromatography ofintermediates.

Provided herein is a method for improving the pharmaceutical andmedicinal behavior of a peptide, thereby improving its duration ofaction, bioavailability or its stability in formulation, comprisingcovalent attachment of a surfactant X to the peptide, wherein:

-   -   X is

-   -   -   wherein        -   A is a hydrophobic group; and        -   B is a hydrophilic group covalently attached to the peptide            via a linker amino acid.

In some embodiments, A is a substituted or unsubstituted C₁-C₃₀ alkylchain, a substituted or unsubstituted alkoxyaryl group or a substitutedor unsubstituted aralkyl group.

In some embodiments, A is a substituted or unsubstituted C₈-C₂₀ alkylchain, a substituted or unsubstituted 1-alkoxyaryl group or asubstituted or unsubstituted 1-aralkyl group.

In some embodiments, A is a substituted or unsubstituted C₁₀-C₂₀ alkylchain, a substituted or unsubstituted 1-alkoxyaryl group or asubstituted or unsubstituted 1-aralkyl group.

In some embodiments, B is a functionalized polyol.

In some embodiments, the polyol is a saccharide,

In some embodiments, the saccharide is a monosaccharide, a disaccharide,or a polysaccharide.

In some embodiments, the saccharide is selected from glucuronic acid,galacturonic acid, diglucuronic acid and maltouronic acid.

The methods of synthesis described above are suitable for synthesis ofall compounds described herein, including compounds of Formula I, II.III, 2-I-1, 2-III, 2-V, 2-VI, 2-VII, 3-I-A, 3-III-A, 3-III-B, or 3-V,and compounds in Table 1, Table 2, Table 3 and Table 4 provided in FIG.1, FIG. 2, FIG. 8 and FIG. 9 respectively.

Provided herein is a method for improving the pharmaceutical andmedicinal behavior of a peptide, thereby improving its duration ofaction, bioavailability or its stability in formulation, comprisingcovalent attachment of a surfactant X to the peptide, wherein:

-   -   X is

-   -   -   wherein        -   A is a substituted or unsubstituted C₁-C₂₀ alkyl chain, a            substituted or unsubstituted alkoxyaryl group or a            substituted or unsubstituted aralkyl group; and        -   B is a saccharide covalently attached to the peptide via a            linker amino acid.

In some embodiments, the surfactant is a 1-alkyl glycoside classsurfactant.

In some embodiments, the hydrophilic group in the surfactant is attachedto the peptide via an amide bond.

In some embodiments, the surfactant is comrosed of1-hexadecyl-beta-D-glucuronic acid, 1-tetradecyl-beta-D-glucuronic acid,1-dodecyl-beta-D-glucuronic acid, 1-decyl-beta-D-glucuronic acid,1-octyl-beta-D-glucuronic acid, 1-hexadecyl-beta-D-diglucuronic acid,1-tetradecyl-beta-D-diglucuronic acid, 1-dodecyl-beta-D-diglucuronicacid, 1-decyl-beta-D-diglucuronic acid, 1-octyl-beta-D-diglucuronicacid.

Provided herein is a method of treating pain in an individual in needthereof comprising administration of a therapeutically effective amountof a peptide product described herein, or a compound of Formula IV to anindividual in need thereof.

Also provided herein is a covalently modified peptide and/or proteinproduct comprising a hydrophilic group as described herein; and ahydrophobic group covalently attached to the hydrophilic group. Inspecific embodiments, the covalently modified peptide and/or proteinproduct comprises a hydrophilic group that is a saccharide and ahydrophobic group that is a C₁-C₂₀ alkyl chain or an aralkyl chain.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Table 1 in FIG. 1 depicts compounds that were prepared bymethods described herein. The specification provides sequences for SEQ.ID. Nos. 1 and 149-169. Additionally, Table 1 of FIG. 1 provides SEQ. IDNumbers for compounds EU-A101 to EU-A199 and EU-A600 to EU-A649 havingSEQ. ID. NOs. 2-148, and SEQ. ID. NO. 645 respectively, as shown inTable 1 of FIG. 1. Compounds in Table 1 of FIG. 1, and their respectiveSEQ. ID. NOs. shown in Table 1 of FIG. 1 are hereby incorporated intothe specification as filed.

FIG. 2. Table 2 in FIG. 2 depicts compounds that were prepared bymethods described herein. The specification provides sequences for SEQ.ID. Nos. 170-174 and SEQ. ID. NOs. 283-302. Additionally, Table 2 ofFIG. 2 provides SEQ. ID. Numbers for compounds EU-201 to EU-299 andEU-900 to EU-908 having SEQ. ID. NOs. 175-282 respectively, as shown inTable 2 of FIG. 2. Compounds in Table 2 of FIG. 2, and their respectiveSEQ. ID. NOs. shown in Table 2 of FIG. 2 are hereby incorporated intothe specification as filed.

FIG. 3. FIG. 3 has two panels. The top panel in FIG. 3 illustrates theinteraction between PTH 1-34 or PTHrP 1-34 and the PTH R1 receptor basedon the x-ray crystal structure (Piozak, A. A., et al. (2009) J Biol Chem284:28382-391) of the extracellular domain of the receptor. The criticalhydrophobic interactions of residues 23′ (W or F), 24′ (L) and 28′ (L orI) are illustrated. The surfactant modification on the modified peptidesdescribed herein, in some instances, replace these critical hydrophobicinteractions. The bottom panel in FIG. 3 shows the structural comparisonbetween the sequence of PTH 1-34 and PTHrP 1-34 and illustrates thestrong regions of identity and homology in the sequences and peptidehelical conformation.

FIG. 4. FIG. 4 shows the cAMP responses of human cells in culture(SaOS2) to stimulation by a representative peptide product describedherein, EU-232. The ordinate (vertical axis) shows the response as apercentage of the maximal response shown to the internal assay standard,i.e., PTHrP. The data illustrates a super-agonistic response to EU-232.

FIG. 5. FIG. 5 shows the response of human cells in culture (SaOS2) totreatment with various doses of EU-285 (a coded sample of human PTHrP).The ordinate (vertical axis) shows the cAMP response as a percentage ofthe maximal response of the internal assay standard, i.e., PTHrP.

FIG. 6. Blood phosphate levels in rat serum were tested at various timepoints after subcutaneous dosing rats with saline (G1), 80 microgramsper kg of PTH (G2), 80 micrograms per kg of EU-232 (G3) or 320micrograms per kg of EU-232 (G4). This surfactant modified analog,EU-232, demonstrates prolonged duration of action, as evidenced by themaximal statistically significant effect, which is seen at the last timepoint in the assay (i.e., 5 hrs post dosing). See Example 6.

FIG. 7. Blood calcium levels in rat serum were tested at various timepoints after subcutaneous dosing in rats with saline (G1), 80 microgramsper kg of PTH (G2), 80 micrograms per kg of EU-232 (G3) or 320micrograms per kg of EU-232 (G4). No groups were statisticallysignificantly different from control (G1). Further, the maximallyeffective dose and time point for EU-232 (G4; at 5 hrs) shows noelevation and thus no indications of a propensity for hypercalcemia at amaximally effective dose. See Example 6.

FIG. 8. Table 3 in FIG. 8 depicts compounds that were prepared bymethods described herein. The specification provides sequences for SEQ.ID. Nos. 303-305 and SEQ. ID. Nos. 619-644. Additionally, Table 3 ofFIG. 8 provides SEQ. ID Numbers for compounds EU-A300 to EU-A425 havingSEQ. ID. NOs. 306-431 respectively, as shown in Table 3 of FIG. 8.Compounds in Table 3 of FIG. 8, and their respective SEQ. ID. NOs. shownin Table 3 of FIG. 8 are hereby incorporated into the specification asfiled.

FIG. 9. Table 4 in FIG. 9 depicts compounds that were prepared bymethods described herein. The specification provides SEQ. ID. Nos.303-305 and SEQ. ID. Nos. 619-644. Additionally, Table 4 of FIG. 9provides SEQ. ID Numbers for compounds EU-A426 to EU-A599 having SEQ.ID. NOs. 432-618 respectively, as shown in Table 4 of FIG. 9.Additionally, Table 4 of FIG. 9 provides SEQ. ID Numbers for compoundsEU-A700 to EU-A1 174 having SEQ. ID. NOs 646-1120. Compounds in Table 4of FIG. 9, and their respective SEQ. ID. NOs. shown in Table 4 of FIG. 9are hereby incorporated into the specification as filed.

FIG. 10. FIG. 10 illustrates the x-ray crystal structure (Runge, S., etal. (2008) J Biol Chem 283: 11340-7) of the binding site of theextracellular domain of the GLP-1 receptor and illustrates criticalhydrophobic binding elements of the receptor and the ligand exendin-4(Val^(19*), Phe^(22*), Trp25*, Leu^(26*)) which are mimicked andreplaced by the hydrophobic 1′-alkyl portion of the surfactant on thepeptides of the invention.

FIG. 11. FIG. 11 shows cellular assay data of a representative compound,EU-A178, acting on cells containing the mu opioid receptor (MOP) andacting as an antagonist on the delta opioid receptor (DOP) incompetition with 30 nM DPDPE. The representative compound shows potent,full agonistic behavior in the MOP agonist assay and highly potentaction as a pure antagonist on the DOP receptor.

FIG. 12. FIG. 12 illustrates the in vivo blood glucose response in db/dbmice upon s.c. administration of the listed amount of test compounds ofthe invention (EU-A993 and EU-A1023) at times t=0, 7 hrs.

FIG. 13. FIG. 13 illustrates examples of the detailed structure of somecompounds of the invention and their linkage through the epsilon aminofunction of a Lys residue, in this case at position 24, to examples ofmono and disaccharide surfactants modified according to one method ofthe invention.

FIG. 14. FIG. 14 illustrates the structure of EU-A937, an example of thetypes of structures of the invention.

FIG. 15. FIG. 15 illustrates the concentrations of compounds EU-A993,EU-A1023 and the native hormone GLP-1 (7-36) with time during incubationin human plasma. This data illustrates the prolonged half life andprotection from proteolysis for the compounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are modified peptides and/or proteins that comprise apeptide and/or protein covalently attached to a hydrophilic group, a“head” (e.g., a polyol, (e.g., a saccharide)); the hydrophilic group iscovalently attached to a hydrophobic group, a “tail”, thereby generatinga surfactant. In some embodiments, use of hydrophobic-linked glycosidesurfactant (e.g., alkyl glycoside) moieties for covalent modification ofthe peptides or proteins, prolongs the duration of action of thepeptides or proteins by multiple mechanisms, including formation ofdepots of the drug at the site of administration in the body and bindingto hydrophobic carrier proteins. In some embodiments, incorporation ofsteric hindrance into peptide and/or protein structure can preventapproach of proteases to the peptide and/or protein product and therebyprevent proteolysis. In some embodiments, surfactant modification (e.g.,covalent attachment of alkyl glycoside class of surfactants) of peptidesand/or proteins as described herein, increases the transport acrossmucosal barriers. Accordingly, the modifications of the peptides and/orproteins described herein provide desirable benefits including and notlimited to, protection from proteolysis, and slowed movement from thesite of administration, thereby leading to prolonged pharmacokineticbehavior (e.g., prolongation of circulating t_(1/2)) and improvedtransmucosal bioavailability.

In some embodiments, interaction of the improved peptides and/orproteins with their receptors is modified in beneficial ways by thetruncation of the sequence, introduction of constraint, and/or theincorporation of steric hindrance. Described herein are novel alkylglycoside reagents that allow for incorporation of both rigidity andsteric hindrance in the modified peptides and/or proteins. In someembodiments, steric hindrance confers receptor selectivity to themodified peptides and/or proteins described herein. In some embodiments,steric hindrance provides protection from proteolysis.

It has been recognized that activation of G Protein-coupled Receptors(GPCRs) cause the activation of multiple second messanger systems.Activated GPCRs also recruit arrestins, which had been thought to beresponsible for stopping GPCR signaling and for internalization of theGPCR. Recently is has been recognized that arrestins themselves are amediator of various signaling pathways, so that ligands which do notrecruit arrestin or which only recruit arrestin can have importantbiological effects through “biased” signaling (Shenoy, S. K. andLefkowitz, R. J. (2011) Trends Pharmacolog Sci 32: 521-533).Incorporation of surfactants into the peptides of the invention, withthe steric hindrance and altered presentation caused by surfactantmodification, may exhibit such surprising and beneficial behavior.Evidence for this behavior is shown by peptides EU-A770 to EU-A773 inexample 5.

Proteins and peptides undergo numerous physical and chemical changesthat may affect potency and safety. Among these are aggregation, whichincludes dimerization, trimerization, and the formation of higher-orderaggregates such as amyloids. Aggregation is a key issue underlyingmultiple potentially deleterious effects for peptide and/orprotein-based therapeutics, including loss of efficacy, alteredpharmacokinetics, reduced stability or product shelf life, and inductionof undesirable immunogenicity. Bioavailability and pharmacokinetics of aself-associating peptide can be influenced by aggregate size and theease of disruption of the non-covalent intermolecular interactions atthe subcutaneous site (Maji, S. K., et al. (2008) PLoS Biol 6: e17). Insome instances peptides can aggregate into subcutaneous depots thatdisassociate with t_(1/2) of 30 or more days. Such slow dissolution canlead to favorable effects such as delivery for one month from a singlesc injection, causes such a low blood concentration that the peptideappears inactive in vivo. Thus hydrophobic aggregation can appear tototally preclude a peptide's bioavailability and effectiveness(Clodfelter, D. K., et al. (1998) Pharm Res 15: 254-262).

Aggregation has been associated with increased immunogenicity of theadministered peptide and/or protein therapeutic. One means to avoid thisproblem is to work with solutions of lower concentration, howeverconcentrated peptide and protein solutions are desirable in someinstances for ease of administration. In some instances, adding polyols(e.g., mono- or oligo-saccharides) or alkyl glycosides to the peptideand/or protein solutions during the course of purification andconcentration, reduces or eliminates aggregation, providing greaterefficiency in the manufacturing process, and providing a final productwhich has less immunogenic potential.

The FDA and other regulatory agencies have increased their scrutiny ofaggregation, especially because of this potential linkage to undesirableimmunogenicity. The immunogenicity of a self-associating peptide can beinfluenced by the formation of aggregates as a result of non-covalentintermolecular interactions. For example, interferon has been shown toaggregate resulting in an antibody response (Hermeling, S., et al.(2006) J Pharm Sci 95: 1084-1096). An antibody response toerythropoietin produced “pure red cell aplasia”, a potentially lifethreatening side effect, in a number of patients receiving recombinantEPO (Casadevall, N., et al. (2002) N Engl J Med 346: 469-475) followinga change in formulation that altered the serum albumin source andconcentration. Insulin loses activity due to protein aggregation uponagitation at temperatures above those found in refrigerated storage(Pezron, I., et al. (2002) J Pharm Sci 91: 1135-1146, Sluzky, V., et al.(1991) Proc Natl Acad Sci USA 88: 9377-9381). Monoclonal antibody basedtherapeutics are subject to inactivation as a result of proteinaggregation (King, H. D., et al. (2002) J Med Chem 45: 4336-4343).Highly concentrated monoclonal antibody formulations pose stability,manufacturing, and delivery challenges related to the protential ofthose antibodies to aggregate. Enzymes may also lose activity as aresult of aggregation. For example thermal inactivation of urokinase isreported to occur via aggregation (Porter, W. R., et al. (1993) ThrombRes 71: 265-279).

Protein stabilization during lyophilization has also posed problems.Protein therapeutics frequently lose biological activity afterlyophilization and reconstitution as a result of aggregate formation andprecipitation. In some instances, addition of reconstitution additives(including, for example, sulfated polysaccharides, polyphosphates, aminoacids, polyethylene glycol (PEG) and various surfactants (Zhang, M. Z.,et al. (1995) Pharm Res 12: 1447-1452, Vrkljan, M., et al. (1994) PharmRes 11: 1004-1008) reduces aggregation. In some cases, a combination ofalcohols, or other organic solvents, is used for solubilization.Trifluoroethanol has an effect on maintaining peptide structure and ithas been used in mixtures to stabilize various peptides (Roccatano, D.,et al. (2002) Proc Natl Acad Sci USA 99: 12179-12184). There is a dangerthat such agents may have a harsh effect on mucosal tissue, causingpatient discomfort or local toxic effects. U.S. Pat. No. 7,390,788 andU.S. Pat. No. 7,425,542 describe the use of alkyl glycosides asstabilizers because of their gentle, non-ionic detergent-like effect.However covalent incorporation of alkyl glycosides into a peptide and/orprotein structure itself has not been described heretofore.

Often naturally occurring oligosaccharides that are covalently attachedto proteins do not have surfactant character. In some embodiments,peptide and/or protein products described herein have a covalentlyattached saccharide and an additional hydrophobic group that conferssurfactant character to the modified peptides, thereby allowing fortunability of bioavailability, immunogenicity, and/or pharmacokineticbehavior of the surfactant-modified peptides.

Proteins and peptides modified with oligosaccharides are described in,for example, Jensen, K. J. and Brask, J. (2005) Biopolymers 80: 747-761,through incorporation of saccharide or oligosaccharide structures usingenzymatic (Gijsen, H. J., et al. (1996) Chem Rev 96: 443-474; Sears, P.and Wong, C. H. (1998) Cell Mol Life Sci 54: 223-252; Guo, Z. and Shao,N. (2005) Med Res Rev 25: 655-678) or chemical approaches (Urge, L., etal. (1992) Biochem Biophys Res Commun 184: 1125-1132; Salvador, L. A.,et al. (1995) Tetrahedron 51: 5643-5656; Kihlberg, J., et al. (1997)Methods Enzymol 289: 221-245; Gregoriadis, G., et al. (2000) Cell MolLife Sci 57: 1964-1969; Chakraborty, T. K., et al. (2005) Glycoconj J22: 83-93; Liu, M., et al. (2005) Carbohydr Res 340: 2111-2122; Payne,R. J., et al. (2007) J Am Chem Soc 129: 13527-13536; Pedersen, S. L., etal. (2010) Chembiochem 11: 366-374). Peptides as well as proteins havebeen modified by glycosylation (Filira, F., et al. (2003) Org BiomolChem 1: 3059-3063); (Negri, L., et al. (1999) J Med Chem 42: 400-404);(Negri, L., et al. (1998) Br J Pharmacol 124: 1516-1522); Rocchi, R., etal. (1987) Int J Pept Protein Res 29: 250-261; Filira, F., et al. (1990)Int J Biol Macromol 12: 41-49; Gobbo, M., et al. (1992) Int J PeptProtein Res 40: 54-61; Urge, L., et al. (1992) Biochem Biophys ResCommun 184: 1125-1132; Djedaini-Pilard, F., et al. (1993) TetrahedronLett 34: 2457-2460; Drouillat, B., et al. (1997) Bioorg Med Chem Lett 7:2247-2250; Lohof, E., et al. (2000) Angew Chem Int Ed Engl 39:2761-2764; Gruner, S. A., et al. (2001) Org Lett 3: 3723-3725; Pean, C.,et al. (2001) Biochim Biophys Acta 1541: 150-160; Filira, F., et al.(2003) Org Biomol Chem 1: 3059-3063; Grotenbreg, G. M., et al. (2004) JOrg Chem 69: 7851-7859; Biondi, L., et al. (2007) J Pept Sci 13:179-189; Koda, Y., et al. (2008) Bioorg Med Chem 16: 6286-6296; LoweryJ. J., et al. (2011) J Pharmacol Exptl Therap 336: 767-78; Yamamoto, T.,et al. (2009) J Med Chem 52: 5164-5175).

However, the aforementioned attempts do not describe an additionalhydrophobic group attached to the peptide-linked oligosaccharide.Accordingly, provided herein are modified peptides and/or proteins thatincorporate a hydrophobic group attached to a saccharide and/oroligosaccharide that is covalently attached to the peptide and/orprotein and that allow for tunability of bioavailability, immunogenicityand pharmacokinetic behaviour. Accordingly, also provided herein aresurfactant reagents comprising an oligosaccharide and a hydrophobicgroup, that allow for modification of peptide and/or proeins.

Provided herein is the use of saccharide-based surfactants in covalentlinkage to a peptide for improvement of peptide and/or proteinproperties. In some embodiments, surfactant modification (e.g., covalentattachment of alkyl glycoside class of surfactants) of peptides and/orproteins as described herein, increases the transport across mucosalbarriers. In some embodiments, covalent attachment of a surfactant to apeptide and/or protein product prevents aggregation of the peptideand/or protein.

The surfactant-modified peptides and/or proteins described hereinovercome limitations of peptide pharmaceuticals including and notlimited to short duration of action, poor bioavailability, aggregation,immunogenicity and lack of receptor subtype specificity through thecovalent incorporation of surfactants such as alkyl glycosides as novelpeptide and protein modifiers.

In certain instances, the effects of surfactants are beneficial withrespect to the physical properties or performance of pharmaceuticalformulations, but are irritating to the skin and/or other tissues and inparticular are irritating to mucosal membranes such as those found inthe nose, mouth, eye, vagina, rectum, buccal or sublingual areas.Additionally, in some instances, surfactants denature proteins thusdestroying their biological function. Since surfactants exert theireffects above the critical micelle concentration (CMC), surfactants withlow CMC's are desirable so that they may be utilized with effectivenessat low concentrations or in small amounts in pharmaceuticalformulations. Accordingly, in some embodiments, surfactants (e.g., alkylglycosides) suitable for peptide modifications described herein have theCMC's less than about 1 mM in pure water or in aqueous solutions. By wayof example only, certain CMC values for alkyl glycosides in water are:Octyl maltoside 19.5 mM; Decyl maltoside 1.8 mM; Dodecyl-β-D-maltoside0.17 mM; Tridecyl maltoside 0.03 mM; Tetradecyl maltoside 0.01 mM;Sucrose dodecanoate 0.3 mM. It will be appreciated that a suitablesurfactant could have a higher or lower CMC depending on the peptideand/or protein that is modified. As used herein, “Critical MicelleConcentration” or “CMC” is the concentration of an amphiphilic component(alkyl glycoside) in solution at which the formation of micelles(spherical micelles, round rods, lamellar structures etc.) in thesolution is initiated. In certain embodiments, the alkyl glycosidesdodecyl, tridecyl and tetradecyl maltoside or glucoside as well assucrose dodecanoate, tridecanoate, and tetradecanoate are possess lowerCMC's and are suitable for peptide and/or protein modificationsdescribed herein.

Opioid Peptides and Analogs

In some embodiments, a peptide therapeutic class amenable to the methodsof peptide modifications described herein is that of the peptideopioids. This class derives from the endogenous peptide opioids whichhave a very broad range of functions in the body, carried out throughbinding to the mu (MOR), delta (DOR), and kappa (KOR) opioid receptors(Schiller, P. W. (2005) AAPS J 7: E560-565). Of most interest is theirrole in modulating and, in particular, suppressing of transmission andperception of pain signals. In the development of such agents thecentral side effects (respiratory suppression, place preferenceindicating reward from self administration) are a major concern, soperipherally acting agents would be attractive (Stein, C., et al. (2009)Brain Res Rev 60: 90-113). Studies from a number of labs have suggestedthat the optimal class of agents will have mu opioid receptor agonismwith the possibility of delta receptor antagonism (Schiller, P. W.(2010) Life Sci 86: 598-603).

Although the endomorphins (Janecka, A., et al. (2007) Curr Med Chem 14:3201-3208) are primarily mu receptor specific, judicious modification ofthe framework can result in molecules with both mu and delta selectivity(Lazarus, L. H. and Okada, Y. (2012) Expert Opin Ther Patents 22: 1-14;Keresztes, A., et al. (2010) ChemMedChem 5: 1176-96). Derived from thedermorphin family are the DALDA class of analogs (Schiller, P. W. (2010)Life Sci 86: 598-603). Also derived from the dermorphin family are theTIPP family of peptides (Schiller, P. W., et al., (1999) Biopolymers51:411-25). Described herein are certain opioid peptides that arecovalently attached to a saccharide group of an alkyl-glycosidesurfactant and have improved pharmaceutical properties.

Some of the exemplary synthetic peptide analogs described herein arederived from endomorphins and some are derived from dermorphin, twoclasses of native peptide opioid sequences. In one aspect, the presentpeptide analogs of the native sequences are endorphin related sequencessuch as illustrated by EU-A101 to EU-A115. In another aspect, thepeptide analogs are dermorphin-related sequences such as EU-A107,EU-A108 and EU-A120 to EU-A133. A related class of opioid peptideanalogs is illustrated by sequences EU-A134 to EU-A142 wherein a Ticresidue replaces the D-Ala residue seen in the dermorphin structure(TIPP family). An additional class of specialized linkage is shown whenTic is replaced by Tic(Ψ[CH2-NH]) as this requires a complementaryresidue Ψ-Phe to complete the linkage.

In some embodiments, a surfactant-modified peptide product has aminoacid sequences corresponding to the general Formula III:

FORMULA III (SEQ. ID. NO. 1) aa₁-aa₂-aa₃-aa₄-aa₅-Z

-   -   wherein:        -   aa₁ is Tyr, Dmt, N-alkyl-Tyr, N-alkyl-Dmt, N-dialkyl-Tyr,            N-dialkyl-Dmt and the like;        -   aa₂ is Pro, D-Arg, D-Cit, D-U(X), D-Ala, Tic,            Tic(Y[CH2-NH]);        -   aa₃ is Phe, Trp, Tmp, D- or L-Nal(1), D- or L-Nal(2),            CaMePhe, Ψ-Phe;        -   aa₄ is Phe, Tmp, D- or L-Nal(1), D- or L-Nal(2), U(X), D- or            L-CαMeU(X);        -   aa₅ is absent or Pro, Aib, U(X), D- or L-CαMeU(X)        -   alkyl or dialkyl is independently a substituted or            unsubstituted C₁-C₁₀ branched or straight chain, or            substituted or unsubstituted aralkyl chain;        -   U is a linking amino acid;        -   X is a functionalized surfactant linked to the side chain of            U;        -   Z is —OH or NH₂.

In some specific embodiments of Formula III, X has the structure:

-   -   wherein:        -   A is a substituted or unsubstituted C₁-C₃₀ alkyl group;        -   R^(1b), R^(1c), and R^(1d) are H;        -   W¹ is —(C═O)—NH—;        -   W² is —O—; and        -   R² is a bond.

In one embodiment, N-alkyl is N-methyl; U is a dibasic amino acid, suchas Lys or Orn; X is a modified nonionic detergent of the 1-alkylglycoside class wherein alkyl is C₁-C₂₀ alkyl or an alkoxyarylsubstituent wherein the glycosidic linkage to the saccharide ring isthrough —O— or other heteroatom (e.g., S or N); Z is NH₂.

In another embodiment, the 1-alkyl group in the 1-alkyl-glycoside issubstituted or unsubstituted C₁-C₁₆alkyl; U is Lys, Z is NH₂;

-   -   aa₁ is Tyr, Dmt, Nα-Me-Tyr, Nα-Me-Dmt;    -   aa₂ is Pro;    -   aa₃ is Phe, Trp, Tmp;    -   aa₄ is Phe, Lys(X);    -   aa₅ is absent or Lys(X).

In a further embodiment, 1-alkyl group in the 1-alkylglycoside issubstituted or unsubstituted C₁-C₂₀alkyl; Z is NH₂;

-   -   aa₁ is Tyr, Dmt;    -   aa₂ is D-Arg, D-Lys(X), Tic, Tic(Ψ[CH2-NH]);    -   aa₃ is Phe, Trp, Tmp, CaMePhe, Ψ-Phe;    -   aa₄ is Phe, Tmp, Lys(X);    -   aa₅ is absent or Pro, Aib, Lys(X), D- or L-CαMeLys(X).

The endomorphin class parent structures are:

-   -   Endomorphin 1—Tyr-Pro-Trp-Phe-NH2    -   Endomorphin 2—Tyr-Pro-Phe-Phe-NH2

Certain analog family substitutions are as shown below:

Position 1 Position 2 Position 3 Position 4 Position 5 Tyr Pro Phe Phe−NH2 Dmt Trp Tmp Lys(X)-NH2 N-Alkyl-Tyr Tmp Lys(X) Aib-NH2 N-Alkyl-DmtCαMePhe CαMeLys(X) Pro-NH2 N-dialkyl-Tyr D- or L-Nal(1) ProN-dialkyl-Dmt D- or L-Nal(2) X = 1-alkyl glucuronyl or 1-alkylmannouronyl substituted with 1-( C₁-C₂₀ alkyl, aryloxyalkyl, and thelike)

Certain sequences contain the following amino acids:

Position 1 Position 2 Position 3 Position 4 Position 5 Tyr Pro Phe Phe—NH2 Dmt Trp Tmp Lys(X)-NH2 Tmp Lys(X) X = 1-alkyl glucuronyl or 1-alkylmannouronyl substituted with 1-( C1-C20 alkyl, aryloxyalkyl, and thelike)

In specific embodiments, analogs with attached surfactants include andare not limited to:

Position Position Position 1 2 3 Position 4 EU-A101 Dmt Pro Tmp Lys(C1-glucuronyl)-NH2 EU-A102 Dmt Pro Tmp Lys (C8-glucuronyl)-NH2 EU-A103Dmt Pro Tmp Lys (C12-glucuronyl)-NH2 EU-A105 Dmt Pro TmpPhe-Lys(C1-glucuronyl)-NH2 EU-A106 Dmt Pro TmpPhe-Lys(C12-glucuronyl)-NH2 EU-A162 Dmt Pro Phe Lys(C1-glucuronyl)-NH2EU-A163 Dmt Pro Phe Lys(C8-glucuronyl)-NH2 EU-A164 Dmt Pro PheLys(C12-glucuronyl)-NH2 EU-A189 Dmt Pro Phe Phe-Lys(C1-glucuronyl)-NH2EU-A190 Dmt Pro Phe Phe-Lys(C12-glucuronyl)-NH2 X = 1-alkyl glucuronylor 1-alkyl mannouronyl substituted with 1-( C₁-C₂₀ alkyl, aryloxyalkyl,and the like)

The Dermorphin class parent structure is:

Parent dermorphin—Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2

Certain analog family structures are as shown below:

Position 1 Position 2 Position 3 Position 4 Tyr D-Arg Phe Phe NH2 DmtD-Lys(X) Trp Tmp Lys(X)-NH2 N-Alkyl-Tyr D-Ala Tmp Lys(X) Pro-NH2N-Alkyl-Dmt Tic CαMePhe CαMeLys(X) Aib-NH2 N-dialkyl-Tyr Tic-(Ψ[CH₂—NH])Ψ-Phe N-dialkyl-Dmt D-or L-Nal(1) D- or L-Nal(1) D- or L-Nal(1) D-orL-Nal(2) D- or L-Nal(2) D- or L-Nal(2) X = 1-alkyl glucuronyl or 1-alkylmannouronyl substituted with 1-( C₁-C₂₀ alkyl, aryloxyalkyl, and thelike)

In some embodiments, certain analogs suitable for attachment ofsurfactants include and are not limited to:

Position Position Position 1 Position 2 3 4 Tyr D-Arg Phe Phe NH2 DmtD-Lys(X) Trp Tmp Lys(X)-NH2 Tic Tmp Lys(X) Pro-NH2 Tic-(Ψ[CH₂—NH]) Ψ-PheAib-NH2 CαMePhe D- or D- or L-Na1(1) L-Na1(1) D- or D- or L-Na1(2)L-Na1(2) X = 1-alkyl glucuronyl or 1-alkyl mannouronyl substituted with1-( C₁-C₂₀ alkyl, aryloxyalkyl, and the like)

In specific embodiments, analogs suitable for attachment of surfactantsinclude and are not limited to:

Position 1 Position 2 Position 3 Position 4 EU-A107 DmtD-Lys(C1-glucuronyl) Tmp Phe-NH2 EU-A108 Dmt D-Lys(C12-glucuronyl) TmpPhe-NH2 EU-A120 Dmt D-Lys(C8-glucuronyl) Nal(1) Phe-NH2 EU-A121 DmtD-Lys(C12-glucuronyl) Nal(1) Phe-NH2 EU-A122 Dmt D-Lys(C16-glucuronyl)Nal(1) Phe-NH2 EU-A123 Dmt D-Lys(C18-glucuronyl) Nal(1) Phe-NH2 EU-A124Dmt D-Lys(C20-glucuronyl) Nal(1) Phe-NH2 EU-A125 DmtD-Lys(C22-glucuronyl) Nal(1) Phe-NH2 EU-A126 Dmt D-Lys(C24-glucuronyl)Nal(1) Phe-NH2 EU-A178 Dmt Tic Phe Lys(C1-glucuronyl)-NH2 EU-A179 DmtTic Phe Lys(C12-glucuronyl)-NH2 EU-A180 Dmt Tic PheLys(C8-glucuronyl)-NH2 EU-A181 Dmt Tic Phe Lys(C10-glucuronyl)-NH2EU-A182 Dmt Tic Phe Lys(C16-glucuronyl)-NH2 EU-A183 Dmt Tic PheLys(C18-glucuronyl)-NH2 EU-A184 Dmt Tic Phe Lys(C20-glucuronyl)-NH2EU-A189 Dmt Tic Phe Phe-Lys(C1-glucuronyl)- NH2 EU-A190 Dmt Tic PhePhe-Lys(C12-glucuronyl)- NH2 EU-A191 Dmt Tic Phe Phe-Lys(C8-glucuronyl)-NH2 EU-A600 Dmt Tic Phe Lys(C1-glucuronyl)-Aib- NH2 EU-A601 Dmt Tic PheLys(C8-glucuronyl)-Aib- NH2 EU-A603 Dmt Tic Phe Lys(C12-glucuronyl)-Aib-NH2 EU-A615 Dmt Tic Phe D-Lys(C1-glucuronyl)-Aib- NH2 EU-A616 Dmt TicPhe D-Lys(C8-glucuronyl)-Aib- NH2 EU-A618 Dmt Tic PheD-Lys(C12-glucuronyl)-NH2 EU-A619 Dmt Tic Phe D-Lys(C16-glucuronyl)-NH2EU-A620 Dmt Tic Phe Lys(C1-glucuronyl)- NHCH₂Ph EU-A621 Dmt Tic PheLys(C8-glucuronyl)- NHCH₂Ph EU-A623 Dmt Tic Phe Lys(C12-glucuronyl)-NHCH₂Ph EU-A624 Dmt Tic Phe Lys(C16-glucuronyl)- NHCH₂Ph EU-A639 Dmt TicPhe D-Lys(C1-glucuronyl)- NHCH₂Ph EU-A642 Dmt Tic PheD-Lys(C12-glucuronyl)- NHCH₂Ph EU-A648 Dmt Tic PheLys(C16-glucuronyl)-NH₂ EU-649 Dmt Tic Phe Lys(C14-glucuronyl)-NH₂ X =1-alkyl glucuronyl or 1-alkyl mannouronyl substituted with 1-(C₁-C₂₀alkyl, aryloxyalkyl, and the like)

Contemplated within the scope of embodiments presented herein arepeptide chains substituted in a suitable position by the substitution ofthe analogs claimed herein by acylation on a linker amino acid, at forexample, the ε-position of Lys, with fatty acids such as octanoic,decanoic, dodecanoic, tetradecanoic, hexadecanoic, octadecanoic,3-phenylpropanoic acids and the like, with saturated or unsaturatedalkyl chains (Nestor, J. J., Jr. (2009) Current Medicinal Chemistry 16:4399-4418; Zhang, L and Bulaj, G. (2012) Curr Med Chem 19: 1602-18).Non-limiting, illustrative examples of such analogs are:

(SEQ. ID. NO. 161) H-Dmt-Tic-Phe-Lys(N-epsilon-acetyl)-NH₂, (SEQ. ID.NO. 162) H-Dmt-Tic-Phe-Lys(N-epsilon-dodecanoyl)-NH₂, (SEQ. ID. NO. 163)H-Dmt-Tic-Phe-Lys(N-epsilon-tetradecanoyl)-NH₂, (SEQ. ID. NO. 164)H-Dmt-Tic-Phe-Lys(N-epsilon-(gamma-glutamyl)-N- alpha-dodecanoyl))-NH₂,(SEQ. ID. NO. 165) H-Dmt-Tic-Phe-Lys(N-epsilon-(gamma-glutamyl)-N-alpha-tetradecanoyl))-NH₂, (SEQ. ID. NO. 166)H-Dmt-Tic-Phe-Lys(N-epsilon-acetyl)-NH-benzyl, (SEQ. ID. NO. 167)H-Dmt-Tic-Phe-Lys(N-epsilon-dodecanoyl)-NH- benzyl, and the like.

In other embodiments of the invention the peptide chain may besubstituted in a suitable position by reaction on a linker amino acid,for example the sulfhydryl of Cys, with a spacer and a hydrophobicmoiety such as a steroid nucleus, for example a cholesterol moiety. Insome of such embodiments, the modified peptide further comprises one ormore PEG chains. Non-limiting examples of such molecules are:

H-Dmt-Tic-Phe-Cys(S-(3-(PEG4-aminoethylacetamide-cholesterol)))-NH₂,(SEQ. ID. NO. 168)

H-Dmt-Tic-Phe-Cys(S-(3-(PEG4-aminoethylacetamide-cholesterol)))-NH-benzyl(SEQ. ID. NO. 169), and the like.

The compounds of Formula I, Formula II, or Formula III are assayed formu opioid receptor activity in a cellular assay (MOP in agonist andantagonist mode), and for delta2 opioid receptor activity in cellularassay (DOP in agonist and antagonist mode) as described in Example 12.

In certain embodiments, as shown in Example 12, a compound having a pureMOP agonist activity along with a pure DOP antagonistic activity, is asuitable profile for clinical applications. Also contemplated within thescope of the disclosure herein are compounds that have low solubilityand low apparent in vitro potency but exhibit prolonged duration ofaction (pharmacodynamic action) in vivo.

Contemplated within the scope of embodiments presented herein arepeptide products of Formula I, Formula II or Formula III, wherein thepeptide product comprises one, or, more than one surfactant groups(e.g., group X having the structure of Formula I). In one embodiment, apeptide product of Formula I, Formula II or Formula III, comprises onesurfactant group. In another embodiment, a peptide product of Formula I,Formula II or Formula III, comprises two surfactant groups. In yetanother embodiment, a peptide product of Formula I, Formula II orFormula III, comprises three surfactant groups.

PTH Peptides and Analogs

Also provided herein, in some embodiments, are reagents andintermediates for synthesis of modified peptides and/or proteins (e.g.,modified PTH, PTHrP, or the like) through the incorporation ofsurfactants.

Provided herein, in some embodiments, are peptide products comprising asurfactant X, covalently attached to a peptide, the peptide comprising alinker amino acid U and at least one other amino acid:

wherein the surfactant X is a group of Formula 2-I:

-   -   wherein:        -   R^(1a) is independently, at each occurrence, a bond, H, a            substituted or unsubstituted C₁-C₃₀ alkyl group, a            substituted or unsubstituted alkoxyaryl group, a substituted            or unsubstituted aralkyl group, or a steroid nucleus            containing moiety;        -   R^(1b), R^(1c), and R^(1d) are each, independently at each            occurrence, a bond, H, a substituted or unsubstituted C₁-C₃₀            alkyl group, a substituted or unsubstituted alkoxyaryl            group, or a substituted or unsubstituted aralkyl group;        -   W¹ is independently, at each occurrence, —CH₂—, —CH₂—O—,            —(C═O), —(C═O)—O—, —(C═O)—NH—, —(C═S)—, —(C═S)—NH—, or            —CH₂—S—;        -   W² is —O—, —CH₂—, or —S—;        -   R² is independently, at each occurrence, a bond, H, a            substituted or unsubstituted C₁-C₃₀ alkyl group, a            substituted or unsubstituted alkoxyaryl group, or a            substituted or unsubstituted aralkyl group, —NH₂, —SH,            C₂-C₄-alkene, C₂-C₄-alkyne, —NH(C═O)—CH₂—Br,            —(CH₂)_(m)-maleimide, or —N₃;        -   n is 1, 2 or 3; and        -   m is 1-10;

the peptide is selected from Formula 2-II:

Formula 2-II (SEQ. ID. NO. 170) aa₁-Val₂-aa₃-Glu₄-aa₅aa₆-aa₇-aa₈-His₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇-aa₁₈-aa₁₉-aa₂₀-aa₂₁-aa₂₂-aa₂₃-aa₂₄-aa₂₅-aa₂₆-Z

-   -   wherein:        -   Z is OH, or —NH—R³,        -   R³ is H, a substituted or unsubstituted C₁-C₁₂ alkyl, or a            PEG chain of less than 10 Da;        -   aa₁ is Aib, Ac5c, or Deg;        -   aa₃ is Aib, Ac4c, or Deg;        -   aa₅ is His, or Ile;        -   aa₆ is Gln, or Cit;        -   aa₇ is Leu, or Phe;        -   aa₈ is Leu, or Nle;        -   aa₁₀ is Asp, Asn, Gln, Glu, Cit, Ala, or Aib;        -   aa₁₁ is Arg, or hArg;        -   aa₁₂ is Gly, Glu, Lys, Ala, Aib, or Ac5c;        -   aa₁₃ is Lys, or Arg;        -   aa₁₄ is Ser, His, Trp, Phe, Leu, Arg, Lys, Glu, or Nal(2);        -   aa₁₅ is Ile, Leu, or Aib;        -   aa₁₆ is Gln, Asn, Glu, Lys, Ser, Cit, Aib, or U;        -   aa₁₇ is Asp, Ser, Aib, Ac4c, Ac5c, or U;        -   aa₁₈ is absent or Leu, Gln, Cit, Aib, Ac5c, Lys, Glu or U;        -   aa₁₉ is absent or Arg, Glu, Aib, Ac4c, Ac5c, or U;        -   aa₂₀ is absent or Arg, Glu, Lys, Aib, Ac4c, Ac5c, or U;        -   aa₂₁ is absent or Arg, Val, Aib, Ac5C, Deg, or U;        -   aa₂₂ is absent or Phe, Glu, Aib, Ac5C, Lys, or U;        -   aa₂₃ is absent or Leu, Phe, Trp, or U;        -   aa₂₄ is absent or His, Arg, or U;        -   aa₂₅ is absent or His, Lys, or U and        -   aa₂₆ is absent or Aib, Ac5c, Lys;        -   U is a natural or unnatural amino acid comprising a            functional group used for covalent attachment to the            surfactant X;    -   wherein any two of aa₁-aa₂₆ are optionally cyclized through        their side chains to form a lactam linkage; and    -   provided that one, or at least one of aa₁₆-aa₂₆ is the linker        amino acid U covalently attached to X.

In some embodiments, n is 1. In some embodiments, n is 2, and a firstglycoside is attached to a second glycoside via a bond between W² of thefirst glycoside and any one of OR^(1b), OR^(1c) or OR^(1d) of the secondglycoside. In some embodiments, n is 3, and a first glycoside isattached to a second glycoside via a bond between W² of the firstglycoside and any one of OR^(1b), OR^(1c) or OR^(1d) of the secondglycoside, and the second glycoside is attached to a third glycoside viaa bond between W² of the second glycoside and any one of OR^(1b),OR^(1c) or OR^(1d) of the third glycoside.

In one embodiment, compounds of Formula I-A are compounds wherein X hasthe structure:

-   -   wherein:        -   R^(1a) is H, a protecting group, a substituted or            unsubstituted C₁-C₃₀ alkyl group, or a steroid nucleus            containing moiety;        -   R^(1b), R^(1c), and R^(1d) are each, independently at each            occurrence, H, a protecting group, or a substituted or            unsubstituted C₁-C₃₀ alkyl group;        -   W¹ is independently, at each occurrence, —CH₂—, —CH₂—O—,            —(C═O), —(C═O)—O—, —(C═O)—NH—, —(C═S)—, —(C═S)—NH—, or            —CH₂—S—;        -   W² is —O— or —S—;        -   R² is a bond, C₂-C₄-alkene, C₂-C₄-alkyne, or            —(CH₂)_(m)-maleimide; and        -   m is 1-10.

In another embodiment, compounds of Formula I-A are compounds wherein Xhas the structure:

Accordingly, in the embodiment described above, R² is a bond.

For instance, in an exemplary embodiment of the structure of X describedabove, W¹ is —C(═O)NH—, R² is a bond between W¹ and an amino acidresidue U within the peptide (e.g., an amino group in the sidechain of alysine residue present in the peptide).

In a further embodiment, compounds of Formula I-A are compounds whereinX has the structure:

For instance, in an exemplary embodiment of the structure of X describedabove, W¹ is —CH₂— and R² is an alkyl-linked maleimide functional groupon X and R² is attached to a suitable moiety of an amino acid residue Uwithin the peptide (e.g., a thiol group in a cysteine residue of thepeptide forms a thioether with the maleimide on X).

In yet another embodiment, compounds of Formula I-A are compoundswherein X has the structure:

-   -   wherein:        -   R^(1a) is H, a protecting group, a substituted or            unsubstituted C₁-C₃₀ alkyl group, or a steroid nucleus            containing moiety;        -   R^(1b), R^(1c), and R^(1d) are each, independently at each            occurrence, H, a protecting group, or a substituted or            unsubstituted C₁-C₃₀ alkyl group;        -   W¹ is —(C═O)—NH—;        -   W² is —O—;        -   R² is a bond.

In an additional embodiment, compounds of Formula I-A are compoundswherein X has the structure:

-   -   wherein:        -   R^(1a) is a substituted or unsubstituted C₁-C₃₀ alkyl group;        -   R^(1b), R^(1c), and R^(1d) are H;        -   W¹ is —(C═O)—NH—;        -   W² is —O—; and        -   R² is a bond.

In some embodiments described above and herein, R^(1a) is a substitutedor unsubstituted C₁-C₃₀ alkyl group.

In some embodiments described above and herein, R^(1a) is a substitutedor unsubstituted C₆-C₂₀ alkyl group.

Also contemplated herein are alternate embodiments wherein X in Formula2-I-A has the structure:

For instance, in an exemplary embodiment of the structure of X describedabove, W¹ is —S—, R² is a C₁-C₃₀ alkyl group, W² is S, R^(1a) is a bondbetween W² and a suitable moiety of an amino acid residue U within thepeptide (e.g., a thiol group in a cysteine residue of the peptide formsa thioether with X).

In another exemplary alternate embodiment of the structure of Xdescribed above, W¹ is —O—, R² is a C₁-C₃₀ alkyl group, W² is O, R^(1a)is a bond between W² and a suitable moiety of an amino acid residue Uwithin the peptide (e.g., a hydroxyl group in a serine or threonineresidue of the peptide forms an ether with X).

In some embodiments, U is used for covalent attachment to X and is adibasic natural or unnatural amino acid, a natural or unnatural aminoacid comprising a thiol, an unnatural amino acid comprising a —N₃ group,an unnatural amino acid comprising an acetylenic group, or an unnaturalamino acid comprising a —NH—C(═O)—CH₂—Br or a —(CH₂)m-maleimide, whereinm is 1-10.

In some embodiments of the peptide product, the surfactant is a 1-alkylglycoside class surfactant. In some embodiments of the peptide product,the surfactant is attached to the peptide via an amide bond.

In some embodiments of the peptide product, the surfactant X iscomprised of 1-eicosyl beta-D-glucuronic acid, 1-octadecylbeta-D-glucuronic acid, 1-hexadecyl beta-D-glucuronic acid,1-tetradecylbeta D-glucuronic acid, 1-dodecyl beta D-glucuronic acid,1-decyl beta-D-glucuronic acid, 1-octyl beta-D-glucuronic acid,1-eicosyl beta-D-diglucuronic acid, 1-octadecyl beta-D-diglucuronicacid, 1-hexadecyl beta-D-diglucuronic acid, 1-tetradecylbeta-D-diglucuronic acid, 1-dodecyl beta-D-diglucuronic acid, 1-decylbeta-D-diglucuronic acid, 1-octyl beta-D-diglucuronic acid, orfunctionalized 1-ecosyl beta-D-glucose, 1-octadecyl beta-D-glucose,1-hexadecyl beta-D-glucose, 1-tetradecyl beta-D-glucose, 1-dodecylbeta-D-glucose, 1-decyl beta-D-glucose, 1-octyl beta-D-glucose,1-eicosyl beta-D-maltoside, 1-octadecyl beta-D-maltoside, 1-hexadecylbeta-D-maltoside, 1-dodecyl beta-D-maltoside, 1-decyl beta-D-maltoside,1-octyl beta-D-maltoside, and the like, and the peptide product isprepared by formation of a linkage between the aforementioned groups anda group on the peptide (e.g., a —COOH group in the aforementioned groupsand an amino group of the peptide).

In some embodiments of the peptide product, U is a terminal amino acidof the peptide. In some embodiments of the peptide product, U is anon-terminal amino acid of the peptide. In some embodiments of thepeptide product, U is a natural D- or L-amino acid. In some embodimentsof the peptide product, U is an unnatural amino acid. In someembodiments of the peptide product, U is selected from Lys, Cys, Orn, oran unnatural amino acid comprising a functional group used for covalentattachment to the surfactant X.

In some embodiments of the peptide product, the functional group usedfor covalent attachment of the peptide to the surfactant X is —NH₂, —SH,—OH, —N₃, haloacetyl, a —(CH₂)_(m)-maleimide (wherein m is 1-10), or anacetylenic group.

In some embodiments side chain functional groups of two different aminoacid residues are linked to form a cyclic lactam. For example a Lys₁₄side chain may form a cyclic lactam with the side chain of Glu₁₈ or aLys₁₈ may form a lactam with the side chain of a Glu₂₂. In someembodiments such lactam structures are reversed and are formed from aGlu₁₄ and a Lys₁₈, for example. Such lactam linkages, in some instances,stabilize alpha helical structures in peptides (Condon, S. M., et al.(2002) Bioorg Med Chem 10: 731-736).

In some embodiments, the peptide product comprising a covalently linkedalkyl glycoside is a covalently modified PTH or analog thereof. In someof such embodiments, the peptide product contains a covalently linked1-O-alkyl β-D-glucuronic acid and the peptide is an analog of PTH.

In some embodiments, a peptide product comprising a covalently linkedalkyl glycoside is a covalently modified PTHrP, or analog thereof. Insome of such embodiments, the peptide product comprises a covalentlylinked 1-O-alkyl β-D-glucuronic acid and the peptide is an analog ofPTHrP.

In some embodiments, the peptide product has the structure of Formula2-III:

Formula 2-III (SEQ. ID. NO. 171)aa₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-aa₁₂-Arg₁₃-aa₁₄-Ile₁₅-aa₁₆-aa₁₇-aa₁₈-aa₁₉-aa₂₀-aa₂₁-aa₂₂-aa₂₃-aa₂₄-aa₂₅-aa₂₆-Z

-   -   wherein:        -   Z is OH or —NH₂;        -   aa₁ is Aib, or Ac5c;        -   aa₁₂ is Ala, Glu, Lys, Aib, or Ac5c;        -   aa₁₄ is Trp, Phe, Lys, Glu or Nal(2);        -   aa₁₆ is Gln, Asn, Glu, Lys, Cit, or U(X);        -   aa₁₇ is Asp, Ser, Aib, Ac4c, Ac5C or U(X);        -   aa₁₈ is absent or Leu, Gln, Aib, Lys, Glu or U(X);        -   aa₁₉ is absent or Arg, Glu, Aib, Ac4c or Ac5c;        -   aa₂₀ is absent or Arg, Glu, Lys, Aib, Ac4c, Ac5c;        -   aa₂₁ is absent or Arg, Val, Aib, Ac5C, or Deg;        -   aa₂₂ is absent or Phe, Glu, Lys or U(X);        -   aa₂₃ is absent or Leu, Phe, Trp or U(X);        -   aa₂₄ is absent or His, Arg, or U(X);        -   aa₂₅ is absent or His, Lys, or U(X); and        -   aa₂₆ is absent or Aib, Ac5c;    -   wherein any two of aa₁-aa₂₆ are optionally cyclized through        their side chains to form a lactam linkage; and    -   provided that one, or at least one of aa₁₆, aa₁₇, aa₁₈, aa₂₂,        aa₂₃, aa₂₄ or aa₂₅ is the linker amino acid U covalently        attached to X.

In some embodiments of Formula 2-III, U is any linker amino aciddescribed herein. In some embodiments, the compound of Formula 2-III isa compound wherein aa₁₂ and aa₁₆ are cyclized through their side chainsto form a lactam linkage. In some embodiments, the compound of Formula2-III is a compound wherein aa₁₆ and aa₂₀ are cyclized through theirside chains to form a lactam linkage.

In some embodiments, the peptide product has the structure:

(SEQ. ID. NO. 180) Ac5c₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-Ala₁₂-Arg₁₃-Trp₁₄-Ile₁₅-Gln₁₆-Aib₁₇-Lys(N-epsilon-1′-dodecyl beta-D-glucuronyl)₁₈-NH₂.

In some embodiments, the peptide product has the structure:

(SEQ. ID. NO. 283) Ac5c₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-Ala₁₂-Arg₁₃-Trp₁₄-Ile₁₅-Gln₁₆-Aib₁₇- Lys(N-epsilon-1′-alkylbeta-D-glucuronyl)₁₈-Aib₁₉- NH₂, wherein alkyl is dodecyl, tetradecyl,hexadecyl, or octadecyl.

In some embodiments, the peptide product has the structure:

(SEQ. ID. NO. 284) Ac5c₁-Val₂-Ai_(b3)-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-Ala₁₂-Arg₁₃-Trp₁₄-Ile₁₅-Gln₁₆-Aib₁₇- Lys(N-epsilon-1′-alkylbeta-D-glucuronyl)₁₈- Ac4c₁₉-NH₂, wherein alkyl is dodecyl, tetradecyl,hexadecyl, or octadecyl.

In some embodiments, the peptide product has the structure:

(SEQ. ID. NO. 285) Ac5_(c1)-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-Ala₁₂-Arg₁₃-Trp₁₄-Ile₁₅-Glu*₁₆-Aib₁₇-Lys(N-epsilon-1′-alkyl beta-D-glucuronyl)₁₈-Aib₁₉- Lys*₂₀-NH₂, whereinGlu*₁₆ and Lys*₂₀ are linked through their sidechains by a lactam andalkyl is dodecyl, tetradecyl, hexadecyl, or octadecyl.

In some embodiments, the peptide product has the structure:

(SEQ. ID. NO. 286) Ac5c₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-Glu*₁₂-Arg₁₃-Trp₁₄-Ile₁₅-Lys*₁₆-Aib₁₇-Lys(N-epsilon-1′-alkyl beta-D-glucuronyl)₁₈- Aib₁₉-NH₂, whereinGlu*₁₂ and Lys*₁₆ are linked through their sidechains by a lactam andalkyl is dodecyl, tetradecyl, hexadecyl, or octadecyl.

In some embodiments, the peptide product has the structure:

(SEQ. ID. NO. 287) Ac5c₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-Ala₁₂-Arg₁₃-Phe₁₄-Ile₁₅-Gln₁₆- Aib₁₇-Lys(N-epsilon-1′-alkylbeta-D-glucuronyl)₁₈- Aib₁₉-NH₂, wherein alkyl is dodecyl, tetradecyl,hexadecyl, or octadecyl.

In some embodiments, the peptide product has the structure:

(SEQ. ID. NO. 288) Ac5c₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-Ala₁₂-Arg₁₃-Trp₁₄-Ile₁₅-Gln₁₆- Aib₁₇-Lys(N-epsilon-1′-alkylbeta-D-glucuronyl)₁₈- Aib₁₉-Aib₂₀-NH₂, wherein alkyl is dodecyl,tetradecyl, hexadecyl, or octadecyl.

In some embodiments, the peptide product has the structure:

(SEQ. ID. NO. 289) Ac5c₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-Glu*₁₂-Arg₁₃-Trp₁₄-Ile₁₅-Lys*₁₆-Aib₁₇-Lys(N-epsilon-1′-alkyl beta-D-glucuronyl)₁₈- Aib₁₉-Aib₂₀-NH₂,wherein Glu*₁₂ and Lys*₁₆ are linked through their sidechains by alactam and alkyl is dodecyl, tetradecyl, hexadecyl, or octadecyl.

In some embodiments, the peptide product has the structure:

(SEQ. ID. NO. 207) Ac5c₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-Ala₁₂-Arg₁₃-Trp₁₄-Ile₁₅-Gln₁₆-Aib₁₇-Lys(N-epsilon-1′-dodecyl beta-D- glucuronyl)₁₈-Aib₁₉-NH₂.

In some embodiments, the peptide product has the structure:

(SEQ. ID. NO. 260) Ac5c₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-Ala₁₂-Arg₁₃-Trp₄-Ile₁₅-Gln₁₆-Aib₁₇-Lys(N-epsilon-1′-tetradecyl beta-D-glucuronyl)₁₈- Aib₁₉-NH₂.

In some embodiments, the peptide product has the structure:

(SEQ. ID. NO. 261) Ac5c₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-Ala₁₂-Arg₁₃-Trp₁₄-Ile₁₅-Gln₁₆-Aib₁₇-Lys(N-epsilon-1′-hexadecyl beta-D-glucuronyl)₁₈- Aib₁₉-NH₂.

In some embodiments, the peptide product has the structure:

(SEQ. ID. NO. 262) Ac5c₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-Ala₁₂-Arg₁₃-Trp₁₄-Ile₁₅-Gln₁₆-Aib₁₇-Lys(N-epsilon-1′-octadecyl beta-D-glucuronyl)₁₈- Aib₁₉-NH_(2.)

In some embodiments, the peptide product has the structure:

(SEQ. ID. NO. 275) Ac5c₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-Ala₁₂-Arg₁₃-Trp₁₄-Ile₁₅-Gln₁₆-Aib₁₇-Lys(N-epsilon-1′-dodecyl beta-D-glucuronyl)₁₈- Aib₁₉-Aib₂₀-NH₂.

In some embodiments, the peptide product has the structure:

(SEQ. ID. NO. 277) Ac5c₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-Ala₁₂-Arg₁₃-Trp₁₄-Ile₁₅-Gln₁₆-Aib₁₇-Lys(N-epsilon-1′-hexadecyl beta-D- glucuronyl)₁₈Aib₁₉-Aib₂₀-NH₂.

In some embodiments, the peptide product is a biologically activepeptide product that binds to the PTH receptor (PTHR1).

In a specific embodiment, the peptide products of Formula 2-I-Adescribed above and herein have the following structure:

wherein R^(1a) is a C₁-C₂₀ alkyl chain as described in Table 2 of FIG.2, R′ is a peptide as described in Table 2 of FIG. 2, W² of Formula I-Ais —O—, and W¹ of Formula 2-I-A is —(C═O)NH— and is part of an amidelinkage to the peptide R′. In some of such embodiments, R^(1a) is aC₆-C₂₀ alkyl chain. In some of such embodiments, R^(1a) is a C₈-C₂₀alkyl chain. In some of such embodiments, R^(1a) is a C₈-C₂₀ alkylchain. In some of such embodiments, R^(1a) is a C₈-C₁₈ alkyl chain. Insome of such embodiments, R^(1a) is a C₈-C₁₆ alkyl chain.

In embodiments described above, an amino moiety of an amino acid and/ora peptide R′ (e.g., an amino group of an amino acid residue such as aLysine, or a lysine within the peptide R′) is used to form a covalentlinkage with a compound of structure:

wherein R^(1a) is a C₁-C₂₀ alkyl chain as described in Table 2 of FIG.2.

In such cases, the amino acid having an amino moiety (e.g., a Lysineresidue within the peptide R′) which is used to form a covalent linkageto the compound A described above, is a linker amino acid U which isattached to a surfactant X having the structure of Formula 2-A.Accordingly, as one example, Lys(C12) of Table 2 of FIG. 2 has thefollowing structure:

Also contemplated within the scope of the embodiments presented hereinare peptide products of Formula 2-I-A derived from maltouronicacid-based surfactants through binding at either or both carboxylic acidfunctions. Thus, as one example, peptides in Table 2 of FIG. 2 comprisea lysine linker amino acid bonded to a maltouronic acid based surfactantX and having a structure:

It will be understood that in one embodiment, compounds of Formula 2-I-Aare prepared by attaching a lysine to a group X, followed by attachmentof additional amino acid residues and/or peptides are attached to thelysine-X compound to obtain compounds of Formula 2-I-A. It will beunderstood that other natural or non-natural amino acids describedherein are also suitable for attachment to the surfactant X and aresuitable for attaching additional amino acid/peptides to obtaincompounds of Formula 2-I-A. It will be understood that in anotherembodiment, compounds of Formula 2-I-A are prepared by attaching a fulllength or partial length peptide to a group X, followed by optionalattachment of additional amino acid residues and/or peptides areattached to obtain compounds of Formula 2-I-A.

In a specific embodiment, provided herein are compounds selected fromcompounds of Table 2 in FIG. 2.

Also provided herein are pharmaceutical compositions comprising atherapeutically effective amount of a peptide product described above,or acceptable salt thereof, and at least one pharmaceutically acceptablecarrier or excipient.

In some embodiments of the pharmaceutical compositions, the carrier isan aqueous-based carrier. In some embodiments of the pharmaceuticalcompositions, the carrier is a nonaqueous-based carrier. In someembodiments of the pharmaceutical compositions, the nonaqueous-basedcarrier is a hydrofluoroalkane-like solvent comprising sub-micronanhydrous α-lactose or other excipients.

Contemplated within the scope of embodiments presented herein is thereaction of an amino acid and/or a peptide comprising a linker aminoacid U bearing a nucleophile, and a group X comprising a leaving groupor a functional group that can be activated to contain a leaving group,for example a carboxylic acid, or any other reacting group, therebyallowing for covalent linkage of the amino acid and/or peptide to asurfactant X via the linker amino acid U to provide a peptide product ofFormula 2-I-A.

Also contemplated within the scope of embodiments presented herein isthe reaction of an amino acid and/or a peptide comprising a linker aminoacid U bearing a leaving group or a functional group that can beactivated to contain a leaving group, for example a carboxylic acid, orany other reacting group, and a group X comprising a nucleophilic group,thereby allowing for covalent linkage of the amino acid and/or peptideto a surfactant X via the linker amino acid U to provide a peptideproduct of Formula 2-I-A.

It will be understood that, in one embodiment, Compounds of Formula2-I-A are prepared by reaction of a linker amino acid U with X, followedby addition of further residues to U to obtain the peptide product ofFormula 2-I-A. It will be understood that in an alternative embodiment,Compounds of Formula 2-I-A are prepared by reaction of a suitablepeptide comprising a linker amino acid U with X, followed by optionaladdition of further residues to U, to obtain the peptide product ofFormula 2-I-A.

Provided herein are methods of treating hypoparathyroidism comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a peptide product described above. In some embodiments, thehypoparathyroidism is associated with bone mass reduction.

Also provided herein are methods of stimulating bone repair or favoringthe engraftment of a bone implant comprising administering to a subjectin need thereof a therapeutically effective amount of a peptide productdescribed above.

Also provided herein is a covalently modified PTH or PTHrP peptide oranalog thereof, comprising a hydrophilic group as described herein; anda hydrophobic group covalently attached to the hydrophilic group. Inspecific embodiments, the covalently modified peptide and/or proteinproduct comprises a hydrophilic group that is a saccharide and ahydrophobic group that is a C₁-C₂₀ alkyl chain or an aralkyl chain.

In one embodiment, provided is a method for chemically modifying amolecule by covalent linkage to a surfactant to increase or sustain thebiological action of the composition or molecule, for example, receptorbinding or enzymatic activity. In some embodiments, the molecule is apeptide. The method additionally can include further modificationcomprising covalent attachment of the molecule in the composition to apolymer such as polyethylene glycol.

In another embodiment, provided is a method of reducing or eliminatingimmunogenicity of a peptide and/or protein drug by covalently linkingthe peptide chain to at least one alkyl glycoside wherein the alkyl hasfrom 1 to 30 carbon atoms.

Also provided is a method of treating hypoparathyroidism, osteoporosis,osteopenia, post-menopausal osteoporosis, Paget's disease,glucocorticoid induced osteoporosis, old age osteoporosis, humoralhypercalcemia, or the like comprising administering a drug compositioncomprising a peptide covalently linked to at least one alkyl glycosideand delivered to a vertebrate, wherein the alkyl has from 1 to 30 carbonatoms, or further in the range of 6 to 16 carbon atoms, and whereincovalent linkage of the alkyl glycoside to the peptide increases thestability, bioavailability and/or duration of action of the drug.

In some embodiments, the covalently modified peptides and/or proteinsare covalently modified PTH or PTHrP, or analogs thereof, which aremodified to improve their pharmaceutical and medical properties bycovalent modification with alkyl glycoside surfactant moieties. Thesesurfactant-modified analogs have increased steric hindrance that hinderproteolysis, slows uptake and slows clearance from the body.

Some studies show that approximately 50 percent of patients sufferingfrom osteoporosis discontinue oral bisphosphonate therapy within thefirst year. Among patients who discontinue these treatments, many do sobecause of side effects including intolerance. Although truncatedrecombinant parathyroid hormone 1-34 (rhPTH1-34) is availablecommercially as a bone anabolic agent (Brixen, K. T., et al. (2004)Basic Clin Pharmacol Toxicol 94: 260-270; Dobnig, H. (2004) Expert OpinPharmacother 5: 1153-1162) as teriparatide (Lilly), poor compliance is amajor problem. More recently an antibody (denosumab, Amgen) against theligand controlling osteoclast function has been approved, but it hassignificant known side effects (serious skin infections, observed casesof osteonecrosis of the jaw and significant suppression of boneremodeling), some of which may have as yet unclear long-term effects.

Bone Structure

The architecture of bone in man is maintained and elaborated by thecoordinated function of osteoclasts, which cause bone resorption, andosteoblasts, which lay down new bone matrix. Bone is an important depotfor storage of calcium (a critical signaling ion) in the body and adecrease in ambient extracellular Ca level causes an increase in PTHsecretion in via the Ca-sensing receptors on the parathyroid cellularmembrane. PTH binds to its receptor (PTHR1), present on osteoblasts cellmembranes, leading to expression of the “ligand of receptor activator ofnuclear factor-κB” (RANKL). RANKL binds to its receptor, RANK, onosteoclasts precursors, stimulating their differentiation andproliferation (Boyce, B. F. and Xing, L. (2007) Arthritis Res Ther 9Suppl 1: S1). This leads to bone resorption, mobilization of calciumfrom the bone. PTH also acts to increase renal tubular Ca reabsorptionand indirectly to enhance intestinal Ca absorption via its stimulatoryaction on renal 1-α cholecalciferol hydroxylase (increasing circulatingcalcitriol). Both actions serve to provide a longer term increase incirculating calcium ion.

The native form of human parathyroid hormone (hPTH) is an 84-amino acidpeptide that plays an important role in the maintenance of Calciumhomeostasis in mammals (Rosen, C. J. and Bilezikian, J. P. (2001) J ClinEndocrinol Metab 86: 957-964). A structurally-related but independenthormone, parathyroid hormone-related protein (PTHrP), plays a paracrinerole, focused on bone growth in local tissue. Both hormones bind to thesame receptor on osteoblasts, PTHR1, and cause activation of multiplesignaling pathways, including that regulated by increased cAMP levels.

However intermittent presence of PTH(1-34) leads only to stimulation ofosteoblasts, lack of RANKL expression, and increased bone density.PTH(1-34) exhibits potent anabolic effects on the skeleton when givenexogenously by intermittent injection. A small group of patientsreceived teriparatide by daily sc injections for 6-24 months (Reeve, J.,et al. (1980) Br Med J 280: 1340-1344) and paired bone biopsies revealedsubstantial increases in iliac trabecular bone volume, with evidence ofnew bone formation and a suggestion that there was a dissociationbetween bone formation and resorption rates. Numerous studies haveconfirmed improvements in bone tissue after daily injections of PTHanalogs (Hodsman, A. B., et al. (2005) Endocr Rev 26: 688-703; Cheng,Z., et al. (2009) J Bone Miner Res 24: 209-220). A review of theliterature supports the observation that architectural improvements dooccur within the skeleton after daily teriparatide injections, incontrast to the skeletal architecture observed after therapy withantiresorptive agents, which act mainly by inhibition of osteoblasticactivity to reduce bone turnover, thus preserving rather than buildingnew bone.

Continuous rather than intermittent administration of exogenousPTH(1-34) results in bone absorption. Thus treatment with infusions ofPTH(1-34) for less than 6 hrs result in bone density increases butinfusion for over 8 hr or longer results in bone resorption (Frolik, C.A., et al. (2003) Bone 33: 372-379). The prolonged administration ofPTH(1-34) causes the expression of RANKL, activating RANK on osteoblastprecursors, thus stimulating their differentiation and proliferation(Boyce, B. F. and Xing, L. (2007) Arthritis Res Ther 9 Suppl 1: Si).This observation has led to the current treatment paradigm, once dailyadministration of PTH(1-34) by subcutaneous injection. More recently,studies with infusion of PTH(1-34) for one day and withdrawal for oneweek showed important gains in bone density (Etoh, M. and Yamaguchi, A.(2010) J Bone Miner Metab, 28: 641-9)). Teriparatide has a Cmax of 10minutes and a half life of 19 minutes (Frolik, C. A., et al. (2003) Bone33: 372-379). A more efficacious PTH analog might be expected to givemore substantive bone density increase.

During toxicology studies with PTH(1-34), and with PTH(1-84), it wasobserved that a substantial percentage of the rats developedosteosarcomas, beginning at around 20 months (Tashjian, A. H., Jr. andGoltzman, D. (2008) J Bone Miner Res 23: 803-811). No treatment-relatedsarcomas are reported in human trials with recombinant PTH(1-34),teriparatide (Forteo®). However, treatment with current therapy islimited to <2 yrs of continuous daily subcutaneous injection.

PTH and PTHrP

In some embodiments, the methods and compositions described hereincomprise the use of PTH and/or PTHrP peptides and/or proteins and/oranalogs thereof. All of the biological activity of intact human PTH(hPTH1-84) resides in the N-terminal sequence; most clinical studieshave used the 34-amino acid peptide hPTH(1-34), known as teriparatide.The first two amino acids are obligatory for biological activity, and itappears that the bone anabolic properties are fully maintained by theforeshortened fragment hPTH(1-31) or its cyclized lactam (Whitfield, J.F. and Morley, P. (1995) Trends Pharmacol Sci 16: 382-386). Morerecently studies of sequences as short as hPTH(1-11) have shown activityand can be further modified to decrease their EC₅₀ to the low nM range(Shimizu, M., et al. (2000) J Biol Chem 275: 21836-21843; Shimizu, N.,et al. (2004) J Bone Miner Res 19: 2078-2086).

Studies of the interaction of the PTH and PTHrP with the PTH1R haveindicated that the ligands each have two binding regions, one in theN-terminal 1-14 region and a second in the C-terminal 15-34 region. The1-14 portion has a more locally ordered structure and interacts with the7-transmembrane region of the receptor while the 15-34 region is alphahelical and interacts with the extracellular, N-terminal extension ofthe receptor. While the N-terminal region of these peptides appears tohave the primary role of receptor activation through this juxtamembraneregion interaction, the C-terminal helical region has important bindinginteractions (FIG. 2.) that give rise to higher potency of the ligand(Gardella, T. J., et al. (1994) Endocrinology 135: 1186-1194; Luck, M.D., et al. (1999) Mol Endocrinol 13: 670-680) through interaction withthe extracellular region of the receptor (Dean, T., et al. (2006) J BiolChem 281: 32485-32495; Potetinova, Z., et al. (2006) Biochemistry 45:11113-11121). This leads to a two domain model of binding that also hasbeen extended to other members of the family of class B GPCRs (Holtmann,M. H., et al. (1995) J Biol Chem 270: 14394-14398; Bergwitz, C., et al.(1996) J Biol Chem 271: 26469-26472; Runge, S., et al. (2003) J BiolChem 278: 28005-28010).

Accordingly, provided herein are covalently modified peptides or peptideanalogs that have a sequence that allows for binding to PTH1R. Covalentmodification of ligands in PTH/PTHrP class relies on the development ofN-terminal ligand sequences that interact with the juxtamembrane portionof the PTHR1 coupled to a C-terminal region that has been truncated andsimplified through the use of a surfactant moiety (e.g., a1-alkyl-glucuronic acid moiety such as the glucose-derived1-alkylglucuronic acid). Since much of the C-terminal region interactioninvolves general hydrophobic interaction with the hydrophobic channel(Pioszak, A. A. and Xu, H. E. (2008) Proc Natl Acad Sci USA 105:5034-5039; Pioszak, A. A., et al. (2009) J Biol Chem 284: 28382-28391)in the extracellular region of the receptor, the covalent modificationsdescribed herein allow for increased binding interaction through lowspecificity interaction.

An example of the improved cellular stimulation by a surfactant modifiedpeptide described herein is illustrated in FIG. 4. Thus substantiallygreater cAMP output (125%; super-agonistic stimulation) is shown bycells stimulated with doses of EU-232 (FIG. 5.) than by the internalstandard, human PTHrP. Similary, a coded sample of human PTHrP (FIG. 6.;EU-285) achieves only 100% of the maximal stimulation of the internalassay standard, human PTHrP. EU-232 is modified with a 1-dodecylβ-D-glucouronic acid moiety in the C-terminal region. Importantly,shorter peptide chains or peptide chains of this size unmodified with a1-dodecyl β-D-glucouronic acid moiety can be expected to show decreasedefficacy.

Similarly, the in vivo response to this surfactant modified analogEU-232 shows high potency and prolonged duration of action (FIG. 7.).Blood phosphate levels were tested at various time points after dosingrats with saline (G1), 80 micrograms per kg of PTH (G2), 80 microgramsper kg of EU-232 (G3) or 320 micrograms per kg of EU-232 (G4). EU-232demonstrates prolonged duration of action in that the maximalstatistically significant effect is seen at the last time point in theassay (5 hrs post dosing) and most animals show an increasing biologicaleffect at that timepoint. In contrast PTH has a half-life of 30 minutesin the body and in a similar in vivo assay has a duration of biologicaleffect of about 1 hr. Thus the duration of effect of thesurfactant-modified compund is remarkably longer than that for thenative hormone.

In some embodiments, the surfactant modified analogs described hereindisplays prolonged duration of action by comparison to the unmodifiedpeptide. In some embodiemtns, the duration of action is longer comparedto the unmodified peptide by 10 mins, 30 mins, 45 mins, 1 h, 1.5 h, 2 h,2.5 h, 3 h, 4 h, 5 h, 5.5 h, 6 h, 6.5 h, 7 h, 7.5 h, 8 h, 8.5 h, 9 h,9.5 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 20 h, 24 h, 36 h, or 48h. In some embodiments, the duration of action is 1.5 times, 2 times,2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7times, 8 times, 9 times, or 10 times longer compared to the theunmodified peptide.

As shown in FIG. 7, Blood calcium levels were tested at various timepoints after dosing rats with saline (G1), 80 micrograms per kg of PTH(G2), 80 micrograms per kg of EU-232 (G3) or 320 micrograms per kg ofEU-232 (G4). No groups were statistically significantly different fromcontrol (G1). Importantly, the maximally effective dose and time pointfor EU-232 (G4; at 5 hrs) shows no elevation and thus no indications ofa propensity for hypercalcemia at a maximally effective dose.Hypercalcemia is an important side effect see following administrationof PTH 1-34 and of potent analogs of PTH, so this modification hasdemonstrated an ability to both increase desirable potency on target aswell as diminished propensity for undesired side effects.

The improvements, described above and in the figures, associated withsurfactant modification to yield the peptides described herein havesignificant implications for their use in medicine. Such molecules aresuitable for use by once daily, or less frequent, administration to giveenhanced biological results compared to treatment with short-actingnative hormones such as PTH (T_(1/2) 30 min by s.c. injection) or PTHrP.Surfactant-modified peptides such as EU-232 may be expected to showgreater biological effect when administered by intranasal insufflationdue to the well-known effects of surfactants on nasal bioavailability.

Accordingly, in some embodiments, surfactant-modified peptide productsdescribed herein reduce the occurrence of proteolytic degradation. Insome embodiments, covalent modification of PTH and/or PTHrP and/oranalogs thereof, allows for decreased cost of production oftherapeutics, and provide favorable pharmaceutical properties due to thepresence of the covalently attached surfactant moiety. In someembodiments, surfactant modified PTH and/or PTHrP described hereinprolong the PK and duration of action (PD) behavior of the resultingligands compared to other known peptide ligands (such as those of Dean,et al. (Dean, T., et al. (2006) J Biol Chem 281: 32485-32495)) that lacksuch covalent modifications. Also contemplated within the scope ofembodiments presented herein is long term and safe administration ofsurfactant modified PTH and/or PTHrP and/or analogs thereof.

In some instances, the N-terminal binding region ends at about residue14 and the helical region encompasses residue 16 onward. Thus a ligandoptimized in the 1-14 region with a 1-alkylglucuronic acid modificationin the 15 onward region will have high specific binding (N-terminus)with high potency (1-alkyl modification). Use of an α-helicalstabilizing substitution (Kaul, R. and Balaram, P. (1999) Bioorg MedChem 7: 105-117) in the C-terminal region leads to higher helicalcontent and higher potency. Commonly used α-helical stabilizers are Alaand the class of 1,1-dialkyl amino acids such as Aib, Ac4c, Ac5c and thelike (see definitions below). Minimization of the PTH structure led toshortened analogs (Shimizu, M., et al. (2000) J Biol Chem 275:21836-21843) wherein constrained α-helical stabilizers led to importantpotency increases. For example, substitution of Aib into position 1 and3 of PTH1-14 and PTH1-11 analogs led to increased potency (Shimizu, N.,et al. (2001) J Biol Chem 276: 49003-49012). Incorporation of morehindered α-helical stabilizers in position 1 lead to further potencyincreases (Shimizu, N., et al. (2004) J Bone Miner Res 19: 2078-2086).Substitution of Aib into various positions of PTH1-34 analogs also ledto improvements in potency, particularly substitutions at positions 12and 13 (Peggion, E., et al. (2003) Biopolymers 68: 437-457). However itwas shown that Aib in positions 1 and 3 of the simple PTH1-11 sequencewas not acceptable (Barazza, A., et al. (2005) J Pept Res 65: 23-35).Thus, in some embodiments, the α-helical content of PTH and/or PTHrP isa determinant of peptide product stability (Marx, U. C., et al. (1995) JBiol Chem 270: 15194-15202; Schievano, E., et al. (2000) Biopolymers 54:429-447).

In some embodiments, the use of a long side chain, such as in theNε-(1′-dodecyl beta-D-glucuronyl)-lysine in covalent peptidemodifications described herein, destabilizes an α-helix. Accordingly,also contemplated within the scope of embodiments presented herein aremodifications that comprise α-helical stabilizers. Thus in someembodiments, surfactant modified peptide products described hereincomprise a helix stabilizer (e.g., in the position just N-Terminaland/or just C-terminal of the surfactant substitution). In someembodiments, an α-helix stabilizer is located at position 12 in the PTHand/or PTHrP chain. By way of example only, the table below describesEU-212 to EU-282 certain surfactant modified peptide products (EU-212 toEU-282) that comprise an α-helix stabilizer.

In one aspect, the peptides that are covalently modified and aresuitable for methods described herein are truncated analogs of PTHrPand/or the related hormone PTH, including and not limited to:

(SEQ. ID. NO. 290) hPTH(1-34): Ser₁-Val₂-Ser₃-Glu₄-Ile₅-Gln₆-Leu₇-Met₈-His₉-Asn₁₀-Leu₁₁-Gly₁₂-Lys₁₃-His₁₄-Leu₁₅-Asn₁₆-Ser₁₇-Met₁₈-Glu₁₉-Arg₂₀-Val₂₁-Glu₂₂-Trp₂₃-Leu₂₄-Arg₂₅-Lys₂₆-Lys₂₇-Leu₂₈-Gln₂₉-Asp₃₀-Val₃₁- His₃₂-Asn₃₃-Phe₃₄-OH;or (SEQ. ID. NO. 291) hPTHrP(1-34): Ala₁-Val₂-Ser₃-Glu₄-His₅-Gln₆-Leu₇-Leu₈-His₉-Asp₁₀-Lys₁₁-Gly₁₂-Lys₁₃-Ser₁₄-Leu₁₅-Gln₁₆-Asp₁₇-Leu₁₈-Arg₁₉-Arg₂₀-Arg₂₁-Phe₂₂-Phe₂₃-Leu₂₄-His₂₅-His₂₆-Leu₂₇-Ile₂₈-Ala₂₉-Glu₃₀-Ile₃₁- His₃₂-Thr₃₃-Ala₃₄-OH

In some embodiments, a peptide product described herein has thestructure of Formula 2-V:

FORMULA 2-V (SEQ. ID. NO. 172) aa₁-aa₂-aa₃-aa₄-aa₅aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇-aa₁₈-aa₁₉-aa₂₀-aa₂₁-aa₂₂-aa₂₃-aa₂₄-aa₂₅-aa₂₆-aa₂₇-aa₂₈-aa₂₉-aa₃₀-aa₃₁-aa₃₂-aa₃₃-aa₃₄-aa₃₅-aa₃₆-Zwherein:

-   -   Z is OH, or —NH—R³, wherein R³ is H or C₁-C₁₂ alkyl; or a PEG        chain of less than 10 Da.    -   aa₁ is Ala, Ser, Val, Pro, Aib, Ac5c, or Deg;    -   aa₂ is Val;    -   aa₃ is Ser, Ala, Aib, Ac4c, or Deg;    -   aa₄ is Glu;    -   aa₅ is His, or Ile;    -   aa₆ is Gln, or Cit;    -   aa₇ is Leu, or Phe;    -   aa₈ is Leu, Met, or Nle;    -   aa₉ is His;    -   aa₁₀ is Asp, Asn, Gln, Glu, Cit, Ala, or Aib;    -   aa₁₁ is Lys, Leu, Ile, Arg, or hArg;    -   aa₁₂ is Gly, Ala, Glu, Lys, Aib, or Ac5c;    -   aa₁₃ is Lys, or Arg;    -   aa₁₄ is Ser, His, Trp, Phe, Leu, Arg, Lys, Glu, Nal(2), or        cyclized to position aa₁₈;    -   aa₁₅ is Be, Leu, Aib;    -   aa₁₆ is Gln, Asn, Glu, Lys, Ser, Cit, Aib, Ac5c, U(X);    -   aa₁₇ is Asp, Ser, Aib, Ac4c, Ac5c, U(X), Z;    -   aa₁₈ is absent, Leu, Gln, Cit, Aib, Ac5c, Lys, Glu, or U(X), or        cyclized to position aa₁₄;    -   aa₁₉ is absent, Arg, Glu, Aib, Ac4c, Ac5c, or U(X);    -   aa₂₀ is absent, Arg, Glu, Lys, Aib, Ac4c, Ac5c, or U(X);    -   aa₂₁ is absent, Arg, Val, Aib, Ac5c, Deg, or U(X);    -   aa₂₂ is absent, Phe, Glu, Aib, Ac5c, Lys, U(X), or cyclized to        position aa₁₈ or aa₂₆;    -   aa₂₃ is absent, or Leu, Phe, Trp, or U(X);    -   aa₂₄ is absent, His, Arg, Leu, Aib, Ac5c or U(X);    -   aa₂₅ is absent, His, Lys, Arg, or U(X);    -   aa₂₆ is absent, His, Lys, Arg, Aib, Ac5c or cyclized to position        aa₂₂;    -   aa₂₇ is absent, Leu, or Lys;    -   aa₂₈ is absent, Be, or Leu;    -   aa₂₉ is absent, Ala, Gln, Cit, or Aib;    -   aa₃₀ is absent, Glu, Asp, or Aib;    -   aa₃₁ is absent, Be, Val, Aib, Ac5C, or U(X);    -   aa₃₂ is absent, His, Aib, Ac5C, or U(X);    -   aa₃₃ is absent, Thr, Asn, Aib, Ac5C, or U(X);    -   aa₃₄ is absent, Ala, Phe, Aib, Ac5C, or U(X);    -   aa₃₅ is absent, Aib, Ac5C, or U(X);    -   aa₃₆ is absent, Aib, Ac5C, or U(X);    -   U is a linking amino acid; and    -   X is a surfactant-linked to the side chain of U;    -   wherein any two of aa₁-aa₃₆ are optionally cyclized through        their side chains to form a lactam linkage; and        provided that one, or least one of aa₁-aa₃₆ is U.

In some embodiments, a peptide product described herein comprisesaa₁-aa₂₀ of Formula V as described above (SEQ. ID. NO. 292). In someembodiments, a peptide product described herein comprises aa₁-aa₁₉ ofFormula V as described above (SEQ. ID. NO. 293).

In specific embodiments, the linking amino acid U, is a diamino acidlike Lys or Orn, X is a modified surfactant from the 1-alkyl glycosideclass linked to U, and Z is OH, or —NH—R³, wherein R³ is H or C₁-C₁₂; ora PEG chain of less than 10 Da.

In some embodiments, a peptide product described herein has thestructure of Formula 2-VI:

Formula 2-VI (SEQ. ID. NO. 173) aa₁-Val₂-aa₃-Glu₄-aa₅aa₆-aa₇-aa₈-His₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇-aa₁₈-aa₁₉-aa₂₀-aa₂₁-aa₂₂-aa₂₃-aa₂₄-aa₂₅-aa₂₆-Zwherein:

-   -   Z is OH, or —NH—R³, wherein R³ is H, C₁-C₁₂ alkyl or a PEG chain        of less than 10 Da;    -   aa₁ is Aib, Ac5c, Deg;    -   aa₃ is Aib, Ac4c, Deg;    -   aa₅ is His, Ile;    -   aa₆ is Gln, Cit;    -   aa₇ is Leu, Phe;    -   aa₈ is Leu, Nle;    -   aa₁₀ is Asp, Asn, Gln, Glu, Cit, Ala, Aib;    -   aa₁₁ is Arg, hArg;    -   aa₁₂ is Gly, Ala, Glu, Lys, Aib, Ac5c;    -   aa₁₃ is Lys, Arg;    -   aa₁₄ is Ser, His, Trp, Phe, Leu, Arg, Lys, Nal(2);    -   aa₁₅ is Ile, Leu, Aib;    -   aa₁₆ is Gln, Asn, Glu, Lys, Ser, Cit, Aib, U(X);    -   aa₁₇ is Asp, Ser, Aib, Ac4c, Ac5c, U(X);    -   aa₁₈ is absent or Leu, Gln, Cit, Aib, Ac5c, Lys, Glu, U(X);    -   aa₁₉ is absent or Arg, Glu, Aib, Ac4c, Ac5c, U(X);    -   aa₂₀ is absent or Arg, Glu, Lys, Aib, Ac4c, Ac5c, U(X);    -   aa₂₁ is absent or Arg, Val, Aib, Ac5C, Deg U(X);    -   aa₂₂ is absent or Phe, Glu, Lys or U(X);    -   aa₂₃ is absent or Leu, Phe, Trp or U(X);    -   aa₂₄ is absent or Leu, His, Arg, or U(X);    -   aa₂₅ is absent or His, Lys, or U(X) and    -   aa₂₆ is absent or Aib, Ac5c;    -   U is a linking amino acid;    -   X is a modified surfactant from the 1-alkyl glycoside class        linked to U, wherein the 1-alkyl group is substituted or        unsubstituted C₁-C₂₀ alkyl or substituted or unsubstituted        C₁-C₂₀ aralkyl;        provided that one, or least one of aa₁-aa₂₆ is U.

In some embodiments, a peptide product described herein has thestructure of Formula 2-VII

Formula 2-VII (SEQ. ID. NO. 174)aa₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-aa₁₂-Arg₁₃-aa₁₄-Ile₁₅-aa₁₆-aa₁₇-aa₁₈-aa₁₉-aa₂₀-aa₂₁-aa₂₂-aa₂₃-aa₂₄-aa₂₅-aa₂₆-Zwherein:

-   -   Z is OH or —NH₂;    -   aa₁ is Aib, Ac5c;    -   aa₁₂ is Ala, Glu, Lys, Aib, Ac5c;    -   aa₁₄ is Trp, Phe, Nal(2);    -   aa₁₆ is Gln, Asn, Glu, Lys, Cit, U(X);    -   aa₁₇ is Asp, Ser, Aib, Ac4c, Ac5c, U(X);    -   aa₁₈ is absent or Leu, Gln, Aib, U(X);    -   aa₁₉ is absent or Arg, Glu, Aib, Ac4c, Ac5c;    -   aa₂₀ is absent or Arg, Glu, Lys, Aib, Ac4c, Ac5c;    -   aa₂₁ is absent or Arg, Val, Aib, Ac5C, Deg;    -   aa₂₂ is absent or Phe, Glu, Lys or U(X);    -   aa₂₃ is absent or Leu, Phe, Trp or U(X);    -   aa₂₄ is absent or His, Arg, or U;    -   aa₂₅ is absent or His, Lys, or U and    -   aa₂₆ is absent or Aib, Ac5c;    -   U is a linking amino acid; and    -   X is a modified surfactant from the 1-alkyl glycoside class        linked to U, wherein the 1-alkyl group is substituted or        unsubstituted C₁-C₂₀ alkyl or substituted or unsubstituted        C₁-C₂₀ aralkyl;        provided that one, or least one of aa₁-aa₂₆ is U.

In some embodiments, the peptide product has the structure of Formula2-III:

Formula 2-III (SEQ. ID. NO. 171)aa₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-aa₁₂-Arg₁₃-aa₁₄-Ile₁₅-aa₁₆-aa₁₇-aa₁₈-aa₁₉-aa₂₀-aa₂₁-aa₂₂-aa₂₃-aa₂₄-aa₂₅-aa₂₆-Zwherein:

-   -   Z is OH or —NH₂;    -   aa₁ is Aib, or Ac5c;    -   aa₁₂ is Ala, Aib, Glu, Lys, or Ac5c;    -   aa₁₄ is Trp, Phe, Lys, Glu or Nal(2);    -   aa₁₆ is Gln, Asn, Glu, Lys, Cit, or U(X);    -   aa₁₇ is Asp, Ser, Aib, Ac4c, Ac5c, or U(X);    -   aa₁₈ is absent or Leu, Gln, Aib, Lys, Glu or U(X);    -   aa₁₉ is absent or Arg, Glu, Aib, Ac4c, or Ac5c;    -   aa₂₀ is absent or Arg, Glu, Lys, Aib, Ac4c, Ac5c;    -   aa₂₁ is absent or Arg, Val, Aib, Ac5C, or Deg;    -   aa₂₂ is absent or Phe, Glu, Lys or U(X);    -   aa₂₃ is absent or Leu, Phe, Trp or U(X);    -   aa₂₄ is absent or His, Arg, or U(X);    -   aa₂₅ is absent or His, Lys, or U(X); and    -   aa₂₆ is absent or Aib, Ac5c;    -   wherein any two of aa₁-aa₂₆ are optionally cyclized through        their side chains to form a lactam linkage; and    -   provided that one, or at least one of aa₁₆, aa₁₇, aa₁₈, aa₂₂,        aa₂₃, aa₂₄ or aa₂₅ is the linker amino acid U covalently        attached to X.

In a specific embodiment of Formula 2-III above, X has the structure:

-   -   wherein:        -   R^(1a) is a substituted or unsubstituted C₁-C₃₀ alkyl group;        -   R^(1b), R^(1c), and R^(1d) are H;        -   W¹ is —(C═O)—NH—;        -   W² is —O—; and        -   R² is a bond.

In some of the embodiments described above, R^(1a) is a C₁-C₂₀ alkylgroup, a C₁-C₁₈ alkyl group, a C₁-C₁₆ alkyl group, or C₁-C₁₂ alkylgroup. In some embodiments of Formula 2-III, U is any linker amino aciddescribed herein.

Contemplated within the scope of embodiments presented herein arepeptide products of Formula 2-I-A, Formula 2-III, Formula 2-V, Formula2-VI or Formula 2-VII, wherein the peptide product comprises one, or,more than one surfactant groups (e.g., group X having the structure ofFormula I). In one embodiment, a peptide product of Formula 2-I-A,Formula 2-III, Formula 2-V, Formula 2-VI or Formula 2-VII comprises onesurfactant group. In another embodiment, a peptide product of Formula2-I-A, Formula 2-III, Formula 2-V, Formula 2-VI or Formula 2-VIIcomprises two surfactant groups. In yet another embodiment, a peptideproduct of Formula 2-I-A, Formula 2-III, Formula 2-V, Formula 2-VI orFormula 2-VII comprises three surfactant groups.

Table 2 in FIG. 2 illustrates certain examples of peptides that aresuitable for covalent linkage with surfactants as described herein.

Recognized herein is the importance of certain portions of SEQ. ID. NO.170 for the treatment of conditions associated with bone loss and/orhyperparathyroidism including, and not limited to, osteoporosis,osteopenia, post-menopausal osteoporosis, Paget's disease,glucocorticoid-induced osteoporosis, inflammatory bone loss, fixation ofimplants, osteonecrosis of the jaw, stem cell proliferation, old ageosteoporosis, humoral hypercalcemia, or the like.

Accordingly, provided herein is a method of treating conditionsassociated with bone loss (e.g., osteoporosis) and/orhyperparathyroidism in an individual in need thereof comprisingadministration of a therapeutically effective amount of a PTH analogcomprising amino acid residues aa₁-aa₁₇ of SEQ. ID. NO. 170 to theindividual in need thereof.

In a further embodiment, provided herein is a method of treatingconditions associated with bone loss (e.g., osteoporosis) and/orhyperparathyroidism in an individual in need thereof comprisingadministration of a therapeutically effective amount of a PTH analogcomprising amino acid residues aa₁-aa₁₈ of SEQ. ID. NO. 170 to theindividual in need thereof.

In another embodiment, provided herein is a method of treatingconditions associated with bone loss (e.g., osteoporosis) and/orhyperparathyroidism in an individual in need thereof comprisingadministration of a therapeutically effective amount of a PTH analogcomprising amino acid residues aa₁-aa₁₉ of SEQ. ID. NO. 170 to theindividual in need thereof.

In another embodiment, provided herein is a method of treatingconditions associated with bone loss (e.g., osteoporosis) and/orhyperparathyroidism in an individual in need thereof comprisingadministration of a therapeutically effective amount of a PTH analogcomprising amino acid residues aa₁-aa₂₀ of SEQ. ID. NO. 170 to theindividual in need thereof.

In an additional embodiment, the administration of the said PTH analogdescribed above causes increase in bone density.

Recognized herein is the importance of certain portions of SEQ. ID. NOs.171, 173, 174, 207, 261 or 262 for the treatment of conditionsassociated with bone loss and/or hyperparathyroidism. including, and notlimited to, osteoporosis, osteopenia, post-menopausal osteoporosis,Paget's disease, glucocorticoid-induced osteoporosis, inflammatory boneloss, fixation of implants, osteonecrosis of the jaw, stem cellproliferation, old age osteoporosis, humoral hypercalcemia, or the like.

Accordingly, provided herein is a method of treating conditionsassociated with bone loss and/or hyperparathyroidism in an individual inneed thereof comprising administration of a therapeutically effectiveamount of a PTH analog comprising amino acid residues aa₁-aa₁₇ of SEQ.ID. NOs. 171, 173, 174, 290, 207, 261, 262 or 291 to the individual inneed thereof.

In a further embodiment, provided herein is a method of treatingconditions associated with bone loss (e.g., osteoporosis) and/orhyperparathyroidism in an individual in need thereof comprisingadministration of a therapeutically effective amount of a PTH analogcomprising amino acid residues aa₁-aa₁₈ of SEQ. ID. NOs, 171, 173, 174,290, 207, 261, 262 or 291 to the individual in need thereof.

In another embodiment, provided herein is a method of treatingconditions associated with bone loss (e.g., osteoporosis) and/orhyperparathyroidism in an individual in need thereof comprisingadministration of a therapeutically effective amount of a PTH analogcomprising amino acid residues aa₁-aa₁₉ of SEQ. ID. NOs. 171, 173, 174,290, 207, 261, 262 or 291 to the individual in need thereof.

In another embodiment, provided herein is a method of treatingconditions associated with bone loss (e.g., osteoporosis) and/orhyperparathyroidism in an individual in need thereof comprisingadministration of a therapeutically effective amount of a PTH analogcomprising amino acid residues aa₁-aa₂₀ of SEQ. ID. NOs. 171, 173, 174,290, 275, 281 or 291 to the individual in need thereof.

In an additional embodiment, the administration of the said PTH analogdescribed above causes increase in bone density.

In any of the embodiments described above, the said PTH analog ismodified with a surfactant X of Formula 2-I:

-   -   wherein:        -   R^(1a) is independently, at each occurrence, a bond, H, a            substituted or unsubstituted C₁-C₃₀ alkyl group, a            substituted or unsubstituted alkoxyaryl group, a substituted            or unsubstituted aralkyl group, or a steroid nucleus            containing moiety;        -   R^(1b), R^(1c), and R^(1d) are each, independently at each            occurrence, a bond, H, a substituted or unsubstituted C₁-C₃₀            alkyl group, a substituted or unsubstituted alkoxyaryl            group, or a substituted or unsubstituted aralkyl group;        -   W¹ is independently, at each occurrence, —CH₂—, —CH₂—O—,            —(C═O), —(C═O)—O—, —(C═O)—NH—, —(C═S)—, —(C═S)—NH—, or            —CH₂—S—;        -   W² is —O—, —CH₂— or —S—;        -   R² is independently, at each occurrence, a bond to U, H, a            substituted or unsubstituted C₁-C₃₀ alkyl group, a            substituted or unsubstituted alkoxyaryl group, or a            substituted or unsubstituted aralkyl group, —NH₂, —SH,            C₂-C₄-alkene, C₂-C₄-alkyne, —NH(C═O)—CH₂—Br,            —(CH₂)_(m)-maleimide, or —N₃;        -   n is 1, 2 or 3; and        -   m is 1-10.

In a specific embodiment, the said PTH analog is modified with asurfactant, X having the structure:

-   -   wherein:        -   R^(1a) is a substituted or unsubstituted C₁-C₃₀ alkyl group;        -   R^(1b), R^(1c), and R^(1d) are H;        -   W¹ is —(C═O)—NH—;        -   W² is —O—; and        -   R² is a bond.

In some of the embodiments described above, R^(1a) is a C₁-C₂₀ alkylgroup, a C₈-C₂₀ alkyl group, C₁₂-C₁₈ alkyl group or C₁₄-C₁₈ alkyl group.

Modifications at the amino or carboxyl terminus may optionally beintroduced into the peptides (e.g., PTH or PTHrP) (Nestor, J. J., Jr.(2009) Current Medicinal Chemistry 16: 4399-4418). For example, thepeptides can be truncated or acylated on the N-terminus to yieldpeptides analogs exhibiting low efficacy, partial agonist and antagonistactivity, as has been seen for some peptides (Gourlet, P., et al. (1998)Eur J Pharmacol 354: 105-111, Gozes, I. and Furman, S. (2003) Curr PharmDes 9: 483-494), the contents of which is incorporated herein byreference). For example, deletion of the first 6 residues of bPTH yieldsantagonistic analogs (Mahaffey, J. E., et al. (1979) J Biol Chem 254:6496-6498; Goldman, M. E., et al. (1988) Endocrinology 123: 2597-2599)and a similar operation on peptides described herein generates potentantagonistic analogs. Other modifications to the N-terminus of peptides,such as deletions or incorporation of D-amino acids such as D-Phe alsocan give potent and long acting agonists or antagonists when substitutedwith the modifications described herein such as long chain alkylglycosides. Such agonists and antagonists also have commercial utilityand are within the scope of contemplated embodiments described herein.

Also contemplated within the scope of embodiments presented herein isN-terminal truncation of PTH (e.g. 7-34 residue analogs) or PTHrPthereby providing inverse agonists (Gardella, T. J., et al. (1996)Endocrinology 137: 3936-3941) or antagonists. In some embodiments,inverse agonists and/or antagonists of PTH and/or PTHrP are useful fortreatment of “humoral hypercalcemia” associated with a wide range oftumors.

Also contemplated within the scope of embodiments described herein aresurfactants covalently attached to peptide analogs, wherein the nativepeptide is modified by acetylation, acylation, PEGylation,ADP-ribosylation, amidation, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-link formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, (Nestor, J. J., Jr. (2007) Comprehensive Medicinal ChemistryII 2: 573-601, Nestor, J. J., Jr. (2009) Current Medicinal Chemistry 16:4399-4418, Creighton, T. E. (1993, Wold, F. (1983) PosttranslationalCovalent Modification of Proteins 1-12, Seifter, S. and Englard, S.(1990) Methods Enzymol 182: 626-646, Rattan, S. I., et al. (1992) Ann NY Acad Sci 663: 48-62).

Also contemplated within the scope of embodiments described herein arepeptides that are branched or cyclic, with or without branching. Cyclic,branched and branched circular peptides result from post-translationalnatural processes and are also made by suitable synthetic methods. Insome embodiments, any peptide product described herein comprises apeptide analog described above that is then covalently attached to analkyl-glycoside surfactant moiety.

Also contemplated within the scope of embodiments presented herein arepeptide chains that are substituted in a suitable position by themodification of the analogs claimed herein, e.g., by acylation on alinker amino acid, at for example the ε-position of Lys, with fattyacids such as octanoic, decanoic, dodecanoic, tetradecanoic,hexadecanoic, octadecanoic, 3-phenylpropanoic acids and the like, orwith saturated or unsaturated alkyl chains (Zhang, L. and Bulaj, G.(2012) Curr Med Chem 19: 1602-1618). Non-limiting, illustrative examplesof such analogs are:

(SEQ. ID. NO. 294)Ac5c₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-Ala₁₂-Arg₁₃-Trp₁₄-Ile₁₅-Gln₁₆-Aib₁₇-Lys(N-epsilon-dodecanoyl)₁₈-Aib₁₉-NH_(2,) (SEQ. ID.NO. 295)Ac5c₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-Ala₁₂-Arg₁₃-Trp₁₄-Ile₁₅-Gln₁₆-Aib₁₇-Lys(N-epsilon-dodecanoyl)₁₈-NH_(2,) (SEQ. ID. NO. 296)Ac5c₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-Ala₁₂-Arg₁₃-Trp₁₄-Ile₁₅-Gln₁₆-Aib₁₇-Lys(N-epsilon-palmitoyl)₁₈-Aib₁₉-NH_(2,) (SEQ. ID. NO.297)Ac5c₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-Ala₁₂-Arg₁₃-Phe₁₄-Ile₁₅-Gln₁₆-Aib₁₇-Lys(N-epsilon-dodecanoyl)₁₈-Aib₁₉-NH_(2,) (SEQ. ID.NO. 298)Ac5c₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-Ala₁₂-Arg₁₃-Trp₁₄-Ile₁₅-Gln₁₆-Aib₁₇-Lys(N-epsilon-tetradecanoyl)₁₈-Aib₁₉-NH_(2,) (SEQ. ID.NO. 299)Ac5c₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-Ala₁₂-Arg₁₃-Trp₁₄-Ile₁₅-Gln₁₆-Aib₁₇-Lys(N-epsilon-tetradecanoyl)₁₈-Aib₁₉-Aib₂₀-NH₂, (SEQ.ID. NO. 300)Ac5c₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-Ala₁₂-Arg₁₃-Trp₁₄-Ile₁₅-Gln₁₆-Aib₁₇-Lys(N-epsilon-(Nalpha-dodecanoyl-L-glutamyl))₁₈-Aib₁₉-NH₂,and the like.

In other embodiments, a peptide chain is substituted in a suitableposition by reaction on a linker amino acid, for example the sulfhydrylof Cys, with a spacer and a hydrophobic moiety such as a steroidnucleus, for example a cholesterol moiety. In some of such embodiments,the modified peptide further comprises one or more PEG chains.Non-limiting examples of such molecules are:

(SEQ. ID. NO. 301) Ac5c₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-Ala₁₂-Arg₁₃-Trp₁₄-Ile₁₅-Gln₁₆-Aib₁₇-Cys(S-(3-(PEG4-aminoethylacetamide- Cholesterol)))₁₈-Aib₁₉-NH_(2,) (SEQ.ID. NO. 302) Ac5c₁-Val₂-Aib₃-Glu₄-Ile₅-Gln₆-Leu₇-Nle₈-His₉-Gln₁₀-hArg₁₁-Ala₁₂-Arg₁₃-Trp₁₄-Ile₁₅-Gln₁₆-Aib₁₇-Cys(S-(3-(PEG4-aminoethylacetamide- Cholesterol)))₁₈-NH₂, and the like.

In some embodiments, the modified peptides described herein displaysimproved protection from proteolysis when compared to the unmodifiedpeptide. In some embodiments, the proteolysis of the modified pepetidesdescribed herein is reduced by 10-90%, 10-20%, 10-50%, 20-80%, 20-40%,20-30%, 30-60%, 30-50%, 30-40%, 10-30%, 15-30%, 15-45%, 50-75%, 60-75%,70-90%, or 80-100% when compared with the unmodified peptide. In someembodiments, the proteolysis of the modified pepetides described hereinis reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100%, greater than 5%, greaterthan 10%, greater than 15%, greater than 20%, greater than 25%, greaterthan 30%, greater than 35%, greater than 40%, greater than 45%, greaterthan 50%, greater than 55%, greater than 60%, greater than 65%, greaterthan 70%, greater than 75%, greater than 80%, greater than 85%, greaterthan 90%, greater than 95%, greater than 98%, at least 2%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 98% when comparedwith the unmodified peptide.

In some embodiments, the modified peptides described herein displayarrestin-biased agonism. In other embodiments, the modified peptidesdescribed herein display arrestin-biased antagonism.

In some embodiments, any of the modified peptides described hereindisplays improved receptor binding potency and/or cellular activationpotency compared to the unmodified peptide. In some embodiments, thereceptor binding potency and/or cellular activation potency of themodified pepetides described herein is increased by 10-90%, 10-20%,10-50%, 20-80%, 20-40%, 20-30%, 30-60%, 30-50%, 30-40%, 10-30%, 15-30%,15-45%, 50-75%, 60-75%, 70-90%, or 80-100% when compared with theunmodified peptide. In some embodiments, the receptor binding potencyand/or cellular activation potency of the modified pepetides describedherein is increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100%, 200%, 300%,400%, 500%, 600%, 700%, 800$, 900%, 1000%, greater than 5%, greater than10%, greater than 15%, greater than 20%, greater than 25%, greater than30%, greater than 35%, greater than 40%, greater than 45%, greater than50%, greater than 55%, greater than 60%, greater than 65%, greater than70%, greater than 75%, greater than 80%, greater than 85%, greater than90%, greater than 95%, greater than 98%, at least 2%, at least 5%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 98% when comparedwith the unmodified peptide.

In some embodiments, any of the modified peptides described hereindisplays improved cellular activation potency compared to the unmodifiedpeptide. In some embodiments, the cellular activation potency of themodified pepetides described herein is increased by 10-90%, 10-20%,10-50%, 20-80%, 20-40%, 20-30%, 30-60%, 30-50%, 30-40%, 10-30%, 15-30%,15-45%, 50-75%, 60-75%, 70-90%, or 80-100% when compared with theunmodified peptide. In some embodiments, the cellular activation potencyof the modified pepetides described herein is increased by 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800$, 900%,1000%, greater than 5%, greater than 10%, greater than 15%, greater than20%, greater than 25%, greater than 30%, greater than 35%, greater than40%, greater than 45%, greater than 50%, greater than 55%, greater than60%, greater than 65%, greater than 70%, greater than 75%, greater than80%, greater than 85%, greater than 90%, greater than 95%, greater than98%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 98% when compared with the unmodified peptide.

GLP Peptides and Analogs

Also provided herein, in some embodiments, are reagents andintermediates for synthesis of modified peptides and/or proteins (e.g.,modified GLP-1, glucagon, analogs of glucagon or GLP-1, or the like)through the incorporation of surfactants.

Provided herein, in some embodiments, are peptide products comprising asurfactant X, covalently attached to a peptide, the peptide comprising alinker amino acid U and at least one other amino acid:

-   -   wherein the surfactant X is a group of Formula I:

-   -   wherein:        -   R^(1a) is independently, at each occurrence, a bond, H, a            substituted or unsubstituted C₁-C₃₀ alkyl group, a            substituted or unsubstituted alkoxyaryl group, or a            substituted or unsubstituted aralkyl group;        -   R^(1b), R^(1c), and R^(1d) are each, independently at each            occurrence, a bond, H, a substituted or unsubstituted C₁-C₃₀            alkyl group, a substituted or unsubstituted alkoxyaryl            group, or a substituted or unsubstituted aralkyl group;        -   W¹ is independently, at each occurrence, —CH₂—, —CH₂—O—,            —(C═O), —(C═O)—O—, —(C═O)—NH—, —(C═S)—, —(C═S)—NH—, or            —CH₂—S—;        -   W² is —O—, —CH₂— or —S—;        -   R² is independently, at each occurrence, a bond, H, a            substituted or unsubstituted C₁-C₃₀ alkyl group, a            substituted or unsubstituted alkoxyaryl group, or a            substituted or unsubstituted aralkyl group, —NH₂, —SH,            C₂-C₄-alkene, C₂-C₄-alkyne, —NH(C═O)—CH₂—Br,            —(CH₂)_(m)-maleimide, or —N₃;        -   n is 1, 2 or 3; and        -   m is 1-10;            the peptide is selected from Formula 3-II:

Formula 3-II (SEQ. ID. NO. 303)aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇-aa₁₈-aa₁₉-aa₂₀-aa₂₁-aa₂₂-aa₂₃-aa₂₄-aa₂₅-aa₂₆-aa₂₇-aa₂₈-aa₂₉-aa₃₀-aa₃₁-aa₃₂-aa₃₃-aa₃₄-aa₃₅-aa₃₆-aa₃₇-Z

-   -   wherein:        -   Z is OH, or —NH—R³, wherein R³ is H or C₁-C₁₂ substituted or            unsubstituted alkyl, or a PEG chain of less than 10 Da;        -   aa₁ is His, N—Ac-His, pGlu-His, or N—R³-His;        -   aa₂ is Ser, Ala, Gly, Aib, Ac4c or Ac5c;        -   aa₃ is Gln, or Cit;        -   aa₄ is Gly, or D-Ala;        -   aa₅ is Thr, or Ser;        -   aa₆ is Phe, Trp, F2Phe, Me2Phe, or Nal2;        -   aa₇ is Thr, or Ser;        -   aa₈ is Ser, or Asp;        -   aa₉ is Asp, or Glu;        -   aa₁₀ is Tyr, Leu, Met, Nal2, Bip, or Bip2EtMeO;        -   aa₁₁ is Ser, Asn, or U;        -   aa₁₂ is Lys, Glu, Ser, Arg, or U;        -   aa₁₃ is absent or Tyr, Gln, Cit, or U;        -   aa₁₄ is absent or Leu, Met, Nle, or U;        -   aa₁₅ is absent or Asp, Glu, or U;        -   aa₁₆ is absent or Ser, Gly, Glu, Aib, Ac5c, Lys, Arg, or U;        -   aa₁₇ is absent or Arg, hArg, Gln, Glu, Cit, Aib, Ac4c, Ac5c,            or U;        -   aa₁₈ is absent or Arg, hArg, Ala, Aib, Ac4c, Ac5c, or U;        -   aa₁₉ is absent or Ala, Val, Aib, Ac4c, Ac5c, or U;        -   aa₂₀ is absent or Gln, Lys, Arg, Cit, Glu, Aib, Ac4c, Ac5c,            or U;        -   aa₂₁ is absent or Asp, Glu, Leu, Aib, Ac4c Ac5c, or U;        -   aa₂₂ is absent or Phe, Trp, Nal2, Aib, Ac4c, Ac5c, or U        -   aa₂₃ is absent or Val, Ile, Aib, Ac4c, Ac5c, or U;        -   aa₂₄ is absent or Gln, Ala, Glu, Cit, or U;        -   aa₂₅ is absent or Trp, Nal2, or U;        -   aa₂₆ is absent or Leu, or U;        -   aa₂₇ is absent or Met, Val, Nle, Lys, or U;        -   aa₂₈ is absent or Asn, Lys, or U;        -   aa₂₉ is absent or Thr, Gly, Aib, Ac4c, Ac5c, or U;        -   aa₃₀ is absent or Lys, Aib, Ac4c, Ac5c, or U;        -   aa₃₁ is absent or Arg, Aib, Ac4c, Ac5c, or U;        -   aa₃₂ is absent or Asn, Aib, Ac4c, Ac5c, or U;        -   aa₃₃ is absent or Arg, Aib, Ac4c, Ac5c, or U;        -   aa₃₄ is absent or Asn, Aib, Ac4c, Ac5c, or U;        -   aa₃₅ is absent or Asn, Aib, Ac4c, Ac5c, or U;        -   aa₃₆ is absent or Ile, Aib, Ac4c, Ac5C, or U;        -   aa₃₆ is absent or Ala, Aib, Ac4c, Ac5C, or U;        -   aa₃₇ absent or U;        -   U is a natural or unnatural amino acid comprising a            functional group used for covalent attachment to the            surfactant X;    -   wherein any two of aa₁-aa₃₇ are optionally cyclized through        their side chains to form a lactam linkage; and    -   provided that one, or at least one of aa₁₁-aa₃₇ is the linker        amino acid U covalently attached to X.

In some embodiments, n is 1. In some embodiments, n is 2, and a firstglycoside is attached to a second glycoside via bond between W² of thefirst glycoside and any one of OR^(1b), OR^(1c) or OR^(1d) of the secondglycoside. In some embodiments, n is 3, and a first glycoside isattached to a second glycoside via bond between W² of the firstglycoside and any one of OR^(1b), OR^(1c) or OR^(1d) of the secondglycoside, and the second glycoside is attached to a third glycoside viabond between W² of the second glycoside and any one of OR^(1b), OR^(1c)or OR^(1d) of the third glycoside.

In one embodiment, compounds of Formula I-A are compounds wherein X hasthe structure:

-   -   wherein:        -   R^(1a) is H, a protecting group, a substituted or            unsubstituted C₁-C₃₀ alkyl group, or a steroid nucleus            containing moiety;        -   R^(1b), R^(1c), and R^(1d) are each, independently at each            occurrence, H, a protecting group, or a substituted or            unsubstituted C₁-C₃₀ alkyl group;        -   W¹ is independently, at each occurrence, —CH₂—, —CH₂—O—,            —(C═O), —(C═O)—O—, —(C═O)—NH—, —(C═S)—, —(C═S)—NH—, or            —CH₂—S—;        -   W² is —O—, —S—;        -   R² is a bond, C₂-C₄-alkene, C₂-C₄-alkyne, or            —(CH₂)_(m)-maleimide; and        -   m is 1-10.

In another embodiment, compounds of Formula I-A are compounds wherein Xhas the structure:

Accordingly, in the embodiment described above, R² is a bond.

For instance, in an exemplary embodiment of the structure of X describedabove, W¹ is —C(═O)NH—, R² is a bond between W¹ and an amino acidresidue U within the peptide (e.g., an amino group in the sidechain of alysine residue present in the peptide).

In a further embodiment, compounds of Formula I-A are compounds whereinX has the structure:

For instance, in an exemplary embodiment of the structure of X describedabove, W¹ is —CH₂— and R² is an alkyl-linked maleimide functional groupon X and R² is attached to a suitable moiety of an amino acid residue Uwithin the peptide (e.g., a thiol group in a cysteine residue of thepeptide forms a thioether with the maleimide on X).

In yet another embodiment, compounds of Formula I-A are compoundswherein X has the structure:

-   -   wherein:        -   R^(1a) is H, a protecting group, a substituted or            unsubstituted C₁-C₃₀ alkyl group, or a steroid nucleus            containing moiety;        -   R^(1b), R^(1c), and R^(1d) are each, independently at each            occurrence, H, a protecting group, or a substituted or            unsubstituted C₁-C₃₀ alkyl group;        -   W¹ is —(C═O)—NH—;        -   W² is —O—;        -   R² is a bond.

In an additional embodiment, compounds of Formula I-A are compoundswherein X has the structure:

-   -   wherein:        -   R^(1a) is a substituted or unsubstituted C₁-C₃₀ alkyl group;        -   R^(1b), R^(1c), and R^(1d) are H;        -   W¹ is —(C═O)—NH—;        -   W² is —O—; and        -   R² is a bond.

In some embodiments described above and herein, R^(1a) is a substitutedor unsubstituted C₁-C₃₀ alkyl group.

In some embodiments described above and herein, R^(1a) is a substitutedor unsubstituted C₆-C₂₀ alkyl group.

Also contemplated herein are alternate embodiments wherein X in Formula3-I-A has the structure:

For instance, in an exemplary embodiment of the structure of X describedabove, W¹ is —S—, R² is a C₁-C₃₀ alkyl group, W² is S, R^(1a) is a bondbetween W² and a suitable moiety of an amino acid residue U within thepeptide (e.g., a thiol group in a cysteine residue of the peptide formsa thioether with X).

In another exemplary embodiment of the structure of X described above,W¹ is —O—, R² is a C₁-C₃₀ alkyl group, W² is O, R^(1a) is a bond betweenW² and a suitable moiety of an amino acid residue U within the peptide(e.g., a hydroxyl group in a serine or threonine residue of the peptideforms an ether with X).

In some embodiments, U is used for covalent attachment to X and is adibasic natural or unnatural amino acid, a natural or unnatural aminoacid comprising a thiol, an unnatural amino acid comprising a —N₃ group,an unnatural amino acid comprising an acetylenic group, or an unnaturalamino acid comprising a —NH—C(═O)—CH₂—Br or a —(CH₂)m-maleimide, whereinm is 1-10.

In some embodiments of the peptide product, the surfactant is a 1-alkylglycoside class surfactant. In some embodiments of the peptide product,the surfactant is attached to the peptide via an amide bond.

In some embodiments of the peptide product, the surfactant X iscomprised of 1-eicosyl beta-D-glucuronic acid, 1-octadecylbeta-D-glucuronic acid, 1-hexadecyl beta-D-glucuronic acid,1-tetradecylbeta D-glucuronic acid, 1-dodecyl beta D-glucuronic acid,1-decyl beta-D-glucuronic acid, 1-octyl beta-D-glucuronic acid,1-eicosyl beta-D-diglucuronic acid, 1-octadecyl beta-D-diglucuronicacid, 1-hexadecyl beta-D-diglucuronic acid, 1-tetradecylbeta-D-diglucuronic acid, 1-dodecyl beta-D-diglucuronic acid, 1-decylbeta-D-diglucuronic acid, 1-octyl beta-D-diglucuronic acid, orfunctionalized 1-ecosyl beta-D-glucose, 1-octadecyl beta-D-glucose,1-hexadecyl beta-D-glucose, 1-tetradecyl beta-D-glucose, 1-dodecylbeta-D-glucose, 1-decyl beta-D-glucose, 1-octyl beta-D-glucose,1-eicosyl beta-D-maltoside, 1-octadecyl beta-D-maltoside, 1-hexadecylbeta-D-maltoside, 1-dodecyl beta-D-maltoside, 1-decyl beta-D-maltoside,1-octyl beta-D-maltoside, and the like, and the peptide product isprepared by formation of a linkage between the aforementioned groups anda group on the peptide (e.g., a —COOH group in the aforementioned groupsand an amino group of the peptide).

In some embodiments of the peptide product, U is a terminal amino acidof the peptide. In some embodiments of the peptide product, U is anon-terminal amino acid of the peptide. In some embodiments of thepeptide product, U is a natural D- or L-amino acid. In some embodimentsof the peptide product, U is an unnatural amino acid. In someembodiments of the peptide product, U is selected from Lys, Cys, Orn, oran unnatural amino acid comprising a functional group used for covalentattachment to the surfactant X.

In some embodiments of the peptide product, the functional group usedfor covalent attachment of the peptide to the surfactant X is —NH₂, —SH,—OH, —N₃, haloacetyl, a —(CH₂)_(m)-maleimide (wherein m is 1-10), or anacetylenic group.

In some embodiments side chain functional groups of two different aminoacid residues are linked to form a cyclic lactam. For example, in someembodiments, a Lys side chain forms a cyclic lactam with the side chainof Glu. In some embodiments such lactam structures are reversed and areformed from a Glu and a Lys. Such lactam linkages in some instances areknown to stabilize alpha helical structures in peptides (Condon, S. M.,et al. (2002) Bioorg Med Chem 10: 731-736; Murage, E. N., et al (2008)Bioorg Med Chem 16: 10106-12); Murage, E. N., et al. (2010) J Med Chem53: 6412-20). In some embodiments cysteine residues may be linkedthrough disulfide formation in order to accomplish a similar form ofconformational restriction and assist in the formation of helicalstructures (Li, Y., et al. (2011) Peptides 32: 1400-1407. In someembodiments side chain functional groups of two different amino acidresidues are linked to form a heterocycle generated through a “clickreaction” between side chain azide and alkyne functional groups in orderto achieve a similar form of conformational restriction and stabilizedhelical conformations (Le Chevalier Isaad A., et al. (2009) J PeptideSci 15: 451-4).

In some embodiments, the peptide product comprising a covalently linkedalkyl glycoside is a covalently modified glucagon or analog thereof. Insome of such embodiments, the peptide product contains a covalentlylinked 1-O-alkyl β-D-glucuronic acid and the peptide is an analog ofglucagon.

In some embodiments, a peptide product comprising a covalently linkedalkyl glycoside is a covalently modified GLP-1, or analog thereof. Insome of such embodiments, the peptide product comprises a covalentlylinked 1-O-alkyl β-D-glucuronic acid and the peptide is an analog ofGLP-1.

In some embodiments, the peptide product of Formula I-A has thestructure of Formula III-A

Formula 3-III-A (SEQ. ID. NO. 304)aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇-aa₁₈-aa₁₉-aa₂₀-aa₂₁-aa₂₂-aa₂₃-aa₂₄-aa₂₅-aa₂₆-aa₂₇-aa₂₈-aa₂₉-Z

-   -   wherein:    -   Z is OH, or —NH—R³, wherein R³ is H, or C₁-C₁₂ substituted or        unsubstituted alkyl, or a PEG chain of less than 10 Da;    -   aa₁ is His, N—Ac-His, pGlu-His, or N—R³-His;    -   aa₂ is Ser, Ala, Gly, Aib, Ac4c, or Ac5c;    -   aa₃ is Gln, or Cit;    -   aa₄ is Gly, or D-Ala;    -   aa₅ is Thr, or Ser;    -   aa₆ is Phe, Trp, F2Phe, Me2Phe, or Nal2;    -   aa₇ is Thr, or Ser;    -   aa₈ is Ser, or Asp;    -   aa₉ is Asp, or Glu;    -   aa₁₀ is Tyr, Leu, Met, Nal2, Bip, or Bip2EtMeO;    -   aa₁₁ is Ser, Asn, or U;    -   aa₁₂ is Lys, Glu, Ser, Arg, or U(X);    -   aa₁₃ is absent or Tyr, Gln, Cit, or U(X);    -   aa₁₄ is absent or Leu, Met, Nle, or U(X);    -   aa₁₅ is absent or Asp, Glu, or U(X);    -   aa₁₆ is absent or Ser, Gly, Glu, Aib, Ac5c, Lys, Arg, or U(X);    -   aa₁₇ is absent or Arg, hArg, Gln, Glu, Cit, Aib, Ac4c, Ac5c, or        U(X);    -   aa₁₈ is absent or Arg, hArg, Ala, Aib, Ac4c, Ac5c, or U(X);    -   aa₁₉ is absent or Ala, Val, Aib, Ac4c, Ac5c, or U(X);    -   aa₂₀ is absent or Gln, Lys, Arg, Cit, Glu, Aib, Ac4c, Ac5c, or        U(X);    -   aa₂₁ is absent or Asp, Glu, Leu, Aib, Ac4c, Ac5c, or U(X);    -   aa₂₂ is absent or Phe, Trp, Nal2, Aib, Ac4c, Ac5c, or U(X);    -   aa₂₃ is absent or Val, Ile, Aib, Ac4c, Ac5c, or U(X);    -   aa₂₄ is absent or Gln, Ala, Glu, Cit, or U(X);    -   aa₂₅ is absent or Trp, Nal2, or U(X);    -   aa₂₆ is absent or Leu, or U(X);    -   aa₂₇ is absent or Met, Val, Nle, Lys, or U(X);    -   aa₂₈ is absent or Asn, Lys, or U(X);    -   aa₂₉ is absent or Thr, Gly, Aib, Ac4c, Ac5c, or U(X);    -   wherein any two of aa₁-aa₂₉ are optionally cyclized through        their side chains to form a lactam linkage; and    -   provided that one, or at least one of aa₁₆, aa₁₇, aa₁₈, aa₁₉,        aa₂₀, aa₂₁, aa₂₂, aa₂₃, aa₂₄, aa₂₅, aa₂₆, aa₂₇, aa₂₈ or aa₂₉ is        the natural or unnatural amino acid U covalently attached to X.

In some embodiments, the peptide product of Formula I-A has thestructure of Formula 3-III-B:

Formula 3-III-B (SEQ. ID. NO. 305)His₁-aa₂-aa₃-Gly₄-Thr₅-aa₆-Thr₇-Ser₈-Asp₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇-aa₁₈-aa₁₉-aa₂₀- aa₂₁-aa₂₂-aa₂₃-Z

wherein:

-   -   Z is OH, or —NH—R³, wherein R³ is H or substituted or        unsubstituted C₁-C₁₂ alkyl; or a PEG chain of less than 10 Da;        -   aa₂ is Ser, Ala, Gly, Aib, Ac4c, or Ac5c;        -   aa₃ is Gln, or Cit;        -   aa₆ is Phe, Trp, F2Phe, Me2Phe, MePhe, or Nal2;        -   aa₁₀ is Tyr, Leu, Met, Nal2, Bip, or Bip2EtMeO;        -   aa₁₁ is Ser, Asn, or U(X);        -   aa₁₂ is Lys, Glu, Ser, or U(X);        -   aa₁₃ is absent or Tyr, Gln, Cit, or U(X);        -   aa₁₄ is absent or Leu, Met, Nle, or U(X);        -   aa₁₅ is absent or Asp, Glu, or U(X);        -   aa₁₆ is absent or Ser, Gly, Glu, Aib, Ac4c, Ac5c, Lys, R, or            U(X);        -   aa₁₇ is absent or Arg, hArg, Gln, Glu, Cit, Aib, Ac4c, Ac5c,            or U(X);        -   aa₁₈ is absent or Arg, hArg, Ala, Aib, Ac4c, Ac5c, or U(X);        -   aa₁₉ is absent or Ala, Val, Aib, Ac4c, Ac5c, or U(X);        -   aa₂₀ is absent or Gln, Lys, Arg, Cit, Glu, Aib, Ac4c, Ac5c,            or U(X);        -   aa₂₁ is absent or Asp, Glu, Leu, Aib, Ac4c, Ac5c, or U(X);        -   aa₂₂ is absent or Phe, Aib, Ac4c, Ac5c, or U(X)        -   aa₂₃ is absent or Val, Ile, Aib, Ac4c, Ac5c, or U(X);    -   wherein any two of aa₁-aa₂₃ are optionally cyclized through        their side chains to form a lactam linkage; and    -   provided that one, or at least one of aa₁₆, aa₁₇, aa₁₈, aa₁₉,        aa₂₀, aa₂₁, aa₂₂, aa₂₃ or aa₂₄ is the natural or unnatural amino        acid U covalently attached to X.

In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V, Uis any linker amino acid described herein.

In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V, Uis any linker amino acid described herein. In some embodiments ofFormula 3-I-A, 3-III-A, 3-III-B or Formula 3-V, aa₁₂ is lysine. In someembodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V, aa₁₄ isleucine.

In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V,aa₁₂ is lysine. In some embodiments of Formula I-A, III-A, III-B orFormula V, aa₁₄ is leucine.

In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V,aa₁₈ is a lysine residue attached to X.

In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V,aa₁₇ is a homo Arginine (hArg) residue.

In some embodiments of Formula I-A, III-A, III-B or Formula V, aa₁₇ is aglycine residue.

In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V,aa₂ is an Aib or Ac4c residue.

In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V,the peptide comprises one or more Aib residues.

In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V,peptide comprises one or more Aib residues at the C-terminus.

In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V,the peptide product has the structure (SEQ. ID. NO. 619):

His₁-aa₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Aib₁₆-aa₁₇-Lys(N- omega-1′-alkylbeta-D-glucuronyl)₁₈-aa₁₉-NH₂;wherein

-   -   aa₂ is Aib or Ac4c;    -   aa₁₇ is Arg, hArg or Gln;    -   aa₁₉ is Aib, Ac4c or Ac5c; and    -   alkyl is a C₈ to C₂₀ linear alkyl chain.

In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V,the peptide product ha the structure (SEQ. ID. NO. 620):

His₁-aa₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Aib₁₆-aa₁₇-Lys(N- omega-1′-alkylbeta-D-glucuronyl)₁₈-aa₁₉-aa₂₀-NH₂;wherein

-   -   aa₂ is Aib or Ac4c,    -   aa₁₇ is Arg, hArg or Gln,    -   aa₁₉ and aa20 are individually Aib, Ac4c or Ac5c; and    -   alkyl is a C₈ to C₂₀ linear alkyl chain.

In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V,the peptide product has the structure (SEQ. ID. NO. 621):

His₁-aa₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-aa₁₆-aa₁₇-Lys(N- omega-1′-alkylbeta-D-glucuronyl)₁₈-aa₁₉-NH₂;wherein

-   -   aa₂ is Aib or Ac4c;    -   aa₁₆ is Aib or Ac4c;    -   aa₁₇ is Arg, hArg or Gln;    -   aa₁₉ is Aib, Ac4c or Ac5c; and    -   alkyl is a C₈ to C₂₀ linear alkyl chain.

In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V,aa₁₆ and aa₂₀ are cyclized to form a lactam linkage.

In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V,the peptide product has the structure: (SEQ. ID. NO. 622)

His₁-aa₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-aa₁₆-aa₁₇ Ala₁₈-Ala₁₉-aa₂₀-Glu₂₁-Phe₂₂-Ile₂₃-Lys(N-omega-1′-alkylbeta-D-glucuronyl)₂₄-Trp₂₅-Leu₂₆-aa₂₇-Asn₂₈- Thr₂₉-NH₂;wherein

-   -   aa₂ is Aib or Ac4c;    -   aa₁₆ and aa₂₀ are each individually either Lys or Glu and are        cyclized through their side chains to form a lactam linkage;    -   aa₁₇ is Arg, hArg or Gln;    -   aa₂₇ is Met or Nle; and    -   alkyl is a C₈-C₂₀ linear alkyl chain.

In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V,the peptide product has the structure (SEQ. ID. NO. 623):

His₁-aa₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-cyclic(Glu₁₆-Gln₁₇-Ala₁₈-Ala₁₉-Lys₂₀)-Glu₂₁-Phe₂₂-Ile₂₃-Lys(N- omega-1′-alkylbeta-D-glucuronyl)₂₄-Trp₂₅-Leu₂₆- Met₂₇-Asn₂₈-aa₂₉-NH₂;wherein aa₂ is Aib or Ac4c, aa29 is Thr, Aib, Ac4c, or Ac5c, and the1′-alkyl group is selected from dodecyl, tetradecyl, hexadecyl, oroctadecyl; and the side chains on the amino acids in position 16 and 20are cyclized to form a side chain lactam.

In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V,aa₁₂ and aa₁₆ are cyclized to form a lactam linkage.

In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V,the peptide product has the structure (SEQ. ID. NO. 624):

His₁-aa₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-aa₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-aa₁₆-aa₁₇-Lys(N- omega-1′-alkylbeta-D-glucuronyl)₁₈-aa₁₉-aa₂₀-NH₂;wherein

-   -   aa2 is Aib or Ac4c;    -   aa₁₂ and aa₁₆ are each individually either Lys or Glu and are        cyclized through their side chains to form a lactam linkage;    -   aa₁₇ is Arg, or hArg;    -   aa₁₉ and aa₂₀ are individually either Aib, Ac4c or Ac5c; and    -   alkyl is a C₈-C₂₀ linear alkyl chain.

In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V,the peptide product has the structure (SEQ. ID. NO. 625):

His₁-Ac4c₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-cyclo(Glu₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Lys₁₆₎-aa₁₇-Lys(N-omega-1′-alkyl beta-D-glucuronyl)₁₈- Aib₁₉-Aib₂₀-NH₂;wherein

-   -   aa₁₂ and aa₁₆ are cyclized through their side chains to form a        lactam linkage;    -   aa₁₇ is Arg or hArg; and    -   alkyl is a C₁₂, C₁₄, C₁₆, or C₁₈ linear alkyl chain.

In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V,the peptide product has the structure (SEQ. ID. NO. 626):

His₁-aa₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-aa₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-aa₁₆-aa₁₇-Lys(N- omega-1′-alkylbeta-D-glucuronyl)₁₈-aa₁₉-aa₂₀-NH₂;

-   -   wherein        -   aa₁₂ and aa₁₆ are each individually either Lys or Glu        -   and aa₁₂ and aa₁₆ are cyclized through their side chains to            form a lactam linkage; aa₁₇ is Arg or hArg; aa₁₉ and aa₂₀            are individually either Aib, Ac4c or Ac5c; and the 1′-alkyl            group is selected from dodecyl, tetradecyl, hexadecyl, or            octadecyl.

In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V,the peptide product has the structure (SEQ. ID. NO. 627):

His₁-aa₂-Gln₃-Gly₄-Thr₅-aa₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Ser₁₆-Aib₁₇-Lys(N- omega-1′-dodecylbeta-D-glucuronyl)₁₈-aa₁₉-NH₂;wherein aa₂ is Aib or Ac4c, aa₆ is Me2Phe, MePhe, or Phe; and aa₁₉ isAib, Ac4c, or Ac5c.

In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V,the peptide product has the structure (SEQ. ID. NO. 628):

His₁-aa₂-Gln₃-Gly₄-Thr₅-aa₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Ser₁₆-aa₁₇-Lys(N- omega-1′-dodecylbeta-D-glucuronyl)₁₈-aa₁₉-aa₂₀- NH₂;wherein aa₂ is Aib or Ac4c, aa₆ is Me2Phe, MePhe, or Phe; aa₁₇ is Arg orhArg, and aa₁₉ or aa₂₀ is Aib, Ac4c, or Ac5c.

In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V,the peptide product has the structure (SEQ. ID. NO. 629):

His₁-Aib₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-cyclo(Glu₁₆-Arg₁₇-Ala₁₈-Ala₁₉-Lys₂₀)-Lys(N-omega-1′-alkylbeta-D-glucuronyl)₂₁-Phe₂₂-aa₂₃-NH₂;wherein aa₂₃ is Aib, Ac4c, or Ac5c and the 1′-alkyl group is selectedfrom dodecyl, tetradecyl, hexadecyl, or octadecyl.

In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V,the peptide product has the structure (SEQ. ID. NO. 630):

His₁-aa₂-Gln₃-Gly₄-Thr₅-aa₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-aa₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-aa₁₆-aa₁₇-aa₁₈-Ala₁₉-aa₂₀-Lys(N-omega-1′-alkyl beta-D-glucuronyl)₂₁- Phe₂₂-aa₂₃-NH₂;

-   -   wherein        -   aa₂ is Aib or Ac4c:        -   aa₆ is Me2Phe, MePhe, or Phe;        -   aa₁₂ and aa₁₆ are each individually either Lys or Glu;        -   and aa₁₆ and aa₂₀ are cyclized through their side chains to            form a lactam linkage;        -   aa₁₇ is Arg, hArg or Gln;        -   aa₁₈ is Aib or Ala;        -   aa₂₃ is Aib, Ac4c, or Ac5c and the 1′-alkyl group is            selected from dodecyl, tetradecyl, hexadecyl, or octadecyl.

In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V,the peptide product has the structure (SEQ. ID. NO. 631):

His₁-aa₂-Gln₃-Gly₄-Thr₅-aa₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-aa₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-aa₁₆-aa₁₇-Lys(N- omega-1′-alkylbeta-D-glucuronyl)₁₈-aa₁₉-aa₂₀-NH₂;

-   -   wherein        -   aa₂ is Aib or Ac4c:        -   aa₆ is Phe;        -   aa₁₂ and aa₁₆ are each individually either Lys or Glu; and            aa₁₂ and aa₁₆ are cyclized through their side chains to form            a lactam linkage;        -   aa₁₇ is Arg or hArg;        -   aa₁₉ is Aib, Ac4c, or Ac5c;        -   aa₂₀ is Aib, Ac4c, or Ac5c and the and the 1′-alkyl group is            selected from dodecyl, tetradecyl, hexadecyl, or octadecyl.

In some embodiments, for any compound of Formula 3-I-A, 3-III-A, 3-III-Bor Formula 3-V, X is comprised of a dodecyl alkyl chain.

In some embodiments, the peptide product is a biologically activepeptide product that binds to the GLP1R and/or to the GLCR.

In a specific embodiment, the peptide products of Formula 3-I-A,3-III-A, 3-III-B or Formula 3-V, described above and herein have thefollowing structure:

wherein R^(1a) is a C₁-C₂₀ alkyl chain as described in Table 1 of FIG.1, R′ is a peptide as described in Table 3 of FIG. 8 and Table 4 of FIG.9, W² of Formula I-A is —O—, and W¹ of Formula I-A is —(C═O)NH— and ispart of an amide linkage to the peptide R′. In some of such embodiments,R^(1a) is a C₆-C₂₀ alkyl chain. In some of such embodiments, R^(1a) is aC₈-C₂₀ alkyl chain. In some of such embodiments, R^(1a) is a C₁₂-C₂₀alkyl chain. In some of such embodiments, R^(1a) is a C₁₂-C₁₆ alkylchain.

In embodiments described above, an amino moiety of an amino acid and/ora peptide R′ (e.g., an amino group of an amino acid residue such as aLysine, or a lysine residue within the peptide R′) is used to form acovalent linkage with a compound of structure:

wherein R^(1a) is a C₁-C₂₀ alkyl chain as described above and in Table 3of FIG. 8 and Table 4 of FIG. 9.

In such cases, the amino acid residue having an amino moiety (e.g., aLysine within the peptide R′) which is used to form a covalent linkageto the compound A described above, is a linker amino acid U which isattached to a surfactant X having the structure of Formula A.Accordingly, as one example, Lys(C12) of Table 3 of FIG. 8 and Table 4of FIG. 9 has the following structure:

Also contemplated within the scope of the embodiments presented hereinare peptide products of Formula 3-I-A derived from maltouronicacid-based surfactants through binding at either or both carboxylic acidfunctions. Thus, as one example, peptides in Table 3 of FIG. 8 and Table4 of FIG. 9 comprise a lysine linker amino acid bonded to a maltouronicacid based surfactant X and having a structure:

It will be understood that in one embodiment, compounds of Formula 3-I-Aare prepared by attaching a lysine to a group X, followed by attachmentof additional amino acid residues and/or peptides are attached to thelysine-X compound to obtain compounds of Formula 3-I-A. It will beunderstood that other natural or non-natural amino acids describedherein are also suitable for attachment to the surfactant X and aresuitable for attaching additional amino acid/peptides to obtaincompounds of Formula 3-I-A. It will be understood that in anotherembodiment, compounds of Formula 3-I-A are prepared by attaching a fulllength or partial length peptide to a group X, followed by optionalattachment of additional amino acid residues and/or peptides areattached to obtain compounds of Formula 3-I-A.

In a specific embodiment, provided herein are compounds selected fromcompounds of Table 3 in FIG. 8 and Table 4 in FIG. 9.

Also provided herein are pharmaceutical compositions comprising atherapeutically effective amount of a peptide product described above,or acceptable salt thereof, and at least one pharmaceutically acceptablecarrier or excipient.

In some embodiments of the pharmaceutical compositions, the carrier isan aqueous-based carrier. In some embodiments of the pharmaceuticalcompositions, the carrier is a nonaqueous-based carrier. In someembodiments of the pharmaceutical compositions, the nonaqueous-basedcarrier is a hydrofluoroalkane-like solvent that may comprise sub-micronanhydrous α-lactose or other excipients.

Contemplated within the scope of embodiments presented herein is thereaction of an amino acid and/or a peptide comprising a linker aminoacid U bearing a nucleophile, and a group X comprising a bearing aleaving group or a functional group that can be activated to contain aleaving group, for example a carboxylic acid, or any other reactinggroup, thereby allowing for covalent linkage of the amino acid and/orpeptide to a surfactant X via the linker amino acid U to provide apeptide product of Formula 3-I-A.

Also contemplated within the scope of embodiments presented herein isthe reaction of an amino acid and/or a peptide comprising a linker aminoacid U bearing a bearing a leaving group or a functional group that canbe activated to contain a leaving group, for example a carboxylic acid,or any other reacting group, and a group X comprising a nucleophilicgroup, thereby allowing for covalent linkage of the amino acid and/orpeptide to a surfactant X via the linker amino acid U to provide apeptide product of Formula I-A.

It will be understood that, in one embodiment, Compounds of Formula3-I-A are prepared by reaction of a linker amino acid U with X, followedby addition of further residues to U to obtain the peptide product ofFormula 3-I-A. It will be understood that in an alternative embodiment,Compounds of Formula 3-I-A are prepared by reaction of a suitablepeptide comprising a linker amino acid U with X, followed by optionaladdition of further residues to U, to obtain the peptide product ofFormula 3-I-A.

Provided herein are methods for treating conditions associated withinsulin resistance comprising administration of a compound of Formula3-I-A, 3-III-A, 3-III-B or Formula 3-V.

Provided herein are methods for treating diabetes, diabetic retinopathy,diabetic neuropathy, diabetic nephropathy, wound healing, insulinresistance, hyperglycemia, hyperinsulinemia, metabolic syndrome,diabetic complications, elevated blood levels of free fatty acids orglycerol, hyperlipidemia, obesity, hypertriglyceridemia,atherosclerosis, acute cardiovascular syndrome, infarction, ischemicreperfusion or hypertension, comprising administering a therapeuticallyeffective amount of a peptide product described above and herein to anindividual in need thereof.

Provided herein are methods of reducing weight gain or inducing weightloss comprising administering to a subject in need thereof atherapeutically effective amount of a peptide product described aboveand herein to an individual in need thereof.

Provided herein are methods for treating mammalian conditionscharacterized by obesity-linked insulin resistance or the metabolicsyndrome comprising administering to a subject in need thereof a weightloss-inducing or insulin-sensitizing amount of a peptide productdescribed above and herein to an individual in need thereof.

In some embodiments, the condition to be treated is the metabolicsyndrome (Syndrome X). In some embodiments, the condition to be treatedis diabetes. In some embodiments, the condition to be treated ishyperlipidemia. In some embodiments, the condition to be treated ishypertension. In some embodiments, the condition to be treated isvascular disease including atherosclerosis, or the systemic inflammationcharacterized by elevated C reactive protein.

In some embodiments of the methods, the effective amount of the peptideproduct for administration is from about 0.1 μg/kg/day to about 100.0μg/kg/day, or from 0.01 μg/kg/day to about 1 mg/kg/day or from 0.1μg/kg/day to about 50 mg/kg/day.

Provided herein are methods of treating the metabolic syndrome, or itscomponent diseases, comprising administering to a subject in needthereof a therapeutically effective amount of a peptide productdescribed above. In some embodiments, the metabolic syndrome conditionhas progressed to diabetes.

Also provided herein is a covalently modified GLCR and/or GLP1R bindingpeptide or analog thereof, comprising a hydrophilic group as describedherein; and a hydrophobic group covalently attached to the hydrophilicgroup. In specific embodiments, the covalently modified peptide and/orprotein product comprises a hydrophilic group that is a saccharide and ahydrophobic group that is a C₁-C₂₀ alkyl chain or an aralkyl chain.

Insulin Resistance

The risks associated with prolonged hyperglycemia include an increasedrisk of microvascular complications, sensory neuropathy, myocardialinfarction, stroke, macrovascular mortality, and all-cause mortality.Type 2 diabetes is also linked causally with obesity, an additionalglobal epidemic. At least $232 billion were spent on treatment andprevention of diabetes worldwide in 2007, with three quarters of thatamount spent in industrialized countries on the treatment of long-termcomplications and on general care, such as efforts to prevent micro andmacrovascular complications. In 2007, estimated indirect costs ofdiabetes (disability, lost productivity, and premature death due todiabetes) to the United States economy were $58 billion.

Obesity leads to insulin resistance, a decreased ability of the cells inthe body to react to insulin stimulation through decreased numbers ofinsulin receptors and a decreased coupling of those receptors tocritical intracellular signaling systems. The obese state further leadsto the “metabolic syndrome”, a constellation of diseases (insulinresistance, hypertension, atherosclerosis, et al.) with very largehealthcare consequences. If insulin resistance is diagnosed earlyenough, overt type 2 diabetes can be prevented or delayed, withlifestyle interventions aimed at reducing calorie intake and body fatand through drug treatment to normalize glycemic control. Despitetreatment guidelines recommending early, aggressive intervention, manypatients fail to reach targets for glycemic control. Many factorscontribute to the failure to manage type 2 diabetes successfullyincluding psychosocial and economic influences and shortcomings in theefficacy, convenience and tolerability profiles of availableantidiabetic drugs. The peptide and/or protein products described hereinare designed to overcome these shortcomings.

Incretin Effect

The “incretin effect” is used to describe the phenomenon whereby aglucose load delivered orally produces a much greater insulin secretionthan the same glucose load administered intravenously. This effect ismediated by at least two incretin hormones secreted by intestinalL-cells. Glucose-dependent insulinotropic polypeptide (GIP) andglucagon-like peptide 1 (GLP-1) were identified as incretins and it isthought that healthy individuals may derive up to 70% of their prandialinsulin secretory response from the incretin effect.

Normally the incretin peptides are secreted as needed, in response toingested nutrients, and have a short plasma half-life due to degradationby dipeptidyl peptidase IV (DPP-4) enzyme. In people with type 2diabetes, pancreatic responsiveness to GLP-1 is impaired, but theinsulin-secretory response can be restored with pharmacologic doses ofhuman GLP-1 (Kieffer, T. J., et al. (1995) Endocrinology 136:3585-3596). In addition, GLP-1 promotes beta-cell neogenesis andpreservation (Aaboe, K., et al. (2008) Diabetes Obes Metab 10:994-1003). GLP-1 has additional beneficial effects such as on cardiacfunction: for example it improves left ventricular function (Sokos, G.G., et al. (2006) J Card Fail 12: 694-699) in human subjects. GLP-1 alsoslows gastric emptying in humans and reduces appetite (Toft-Nielsen, M.B., et al. (1999) Diabetes Care 22: 1137-1143).

Treatment of diabetes patients with metabolically stable and long-actinganalogs of GLP-1 is described in, for example, Drab, S. R. (2010)Pharmacotherapy 30: 609-624, suffers from issues related to convenienceof use and side effects such as nausea, risk of pancreatitis and thyroidcarcinoma. GLP-1 analogs provide glucose-dependent stimulation ofinsulin secretion and lead to a reduced risk of hypoglycemia. Inaddition, while a number of the current treatments for diabetes causeweight gain, as described below, GLP-1 analogs induce satiety and a mildweight loss. Accordingly, in some embodiments, provided herein are GLP-1analogs that are long acting and are administered at low doses therebyreducing side-effects associated with current treatments.

A number of peptide gut hormones are known to modulate appetite (Sanger,G. J. and Lee, K. (2008) Nat Rev Drug Discov 7: 241-254). Severalpeptides are derived from tissue-specific, enzymatic processing(prohormone convertases; PCs) of the preproglucagon gene product: e.g.glucagon, GLP-1, glucagon-like peptide-2 (GLP-2), glicentin andoxyntomodulin (OXM) (Drucker, D. J. (2005) Nat Clin Pract EndocrinolMetab 1: 22-31; Sinclair, E. M. and Drucker, D. J. (2005) Physiology(Bethesda) 20: 357-365). GLP-1, GLP-2, glicentin and OXM are co-secretedfrom L-cells in the gut in response to feeding. Preproglucagon isalternatively processed (PC2) to produce glucagon in the alpha cells inthe pancreatic islets. The structure of OXM is essentially glucagon witha C-terminal extension of 8 residues.

In addition to the stimulation of insulin biosynthesis and ofglucose-dependent insulin secretion, GLP-1 and its stable mimetics (e.g.Byetta) also cause modest weight loss in animal models (Mack, C. M., etal. (2006) Int J Obes (Lond) 30: 1332-1340) and in Type 2 diabeticpatients (DeFronzo, R. A., et al. (2005) Diabetes Care 28: 1092-1100;Buse, J. B., et al. (2010) Diabetes Care 33: 1255-1261). Glucagoninfusion reduces food intake in man (Geary, N., et al. (1992) Am JPhysiol 262: R975-980), while continuous glucagon treatment of adiposetissue also promotes lipolysis (Heckemeyer, C. M., et al. (1983)Endocrinology 113: 270-276) and weight loss (Salter, J. M., et al.(1960) Metabolism 9: 753-768; Chan, E. K., et al. (1984) Exp Mol Pathol40: 320-327). Glucagon has wide-ranging effects on energy metabolism(Heppner, K. M., et al. (2010) Physiol Behav)). Glucagon, or analogs,can be used in a diagnostic mode for temporary paralysis of theintestinal tract. Thus at least two of the products from PC processingof the preproglucagon protein are linked to satiety and metaboliceffects.

In rodents, repeated intraperitoneal administration of OXM, a thirdproduct of preproglucagon, has been associated with reduced whiteadipose tissue and a reduction in weight compared with controls (Dakin,C. L., et al. (2004) Endocrinology 145: 2687-2695). Oxm reduced foodintake by 19.3% during an intravenous infusion administration tonormal-weight humans and this effect continues for more than 12 hr.after infusion (Cohen, M. A., et al. (2003) J Clin Endocrinol Metab 88:4696-4701). Treatment of volunteers over a 4 week period resulted in asustained satiety effect and weight loss reflecting a decrease in bodyfat (Wynne, K., et al. (2005) Diabetes 54: 2390-2395).

OXM is structurally homologous with GLP-1 and glucagon, and activatesboth the glucagon receptor (GCGR) and the GLP-1 receptor (GLP1R), butwith 10 to 100 fold less potency than the eponymous ligands. Inaddition, study of OXM interactions with GLP1R suggest it might havedifferent effects on beta-arrestin recruitment compared to GLP-1(Jorgensen, R., et al. (2007) J Pharmacol Exp Ther 322: 148-154), thusacting as a “biased” ligand. A unique receptor for OXM was sought for anumber of years, but has not yet been elucidated and it is assumed toact through the GLP1R and GCGR pathways. Accordingly, provided hereinare methods for surfactant modification of gut peptides that allow forinduction of satiety, weight loss, alleviation of insulin resistanceand/or delay in progression of pre-diabetes to diabetes.

GLP-1

In view of the complex and interacting behavior of the products of thepreproglucagon protein on satiety and metabolism described above,workers from multiple groups have studied the structure activityrelationships on GLP-1 and glucagon structure. Residues throughout thesequences were shown to accept replacement. For example, replacement byAla is well accepted in the N-terminal region of GLP-1, especially at 2,3, 5, 8, 11, and 12 (Adelhorst, K., et al. (1994) J Biol Chem 269:6275-6278).

It was shown that chimeric analogs with the ability to bind to GLP1R andGLCR could be achieved by grafting C-terminal residues from GLP-1 ontothe N-terminus of glucagon (Hjorth, S. A., et al. (1994) J Biol Chem269: 30121-30124). The residue at position 3 (acidic Glu in GLP1 orneutral Gln in Glucagon or OXM) reduces the affinity of glucagon (Runge,S., et al. (2003) J Biol Chem 278: 28005-28010) or OXM (Pocai, A., etal. (2009) Diabetes 58: 2258-2266) for the GlP1R. The effect onmetabolic profile of animals treated with stabilized analogs of GLP-1 orglucagon or OXM with Gln in position 3 was studied (Day, J. W., et al.(2009) Nat Chem Biol 5: 749-757; Druce, M. R., et al. (2009)Endocrinology 150: 1712-1722; Pocai, A., et al. (2009) Diabetes 58:2258-2266). These analogs were designed to have agonistic action on bothGLP1R and on GCGR (Day, J. W., et al. US 2010/0190701 A1).

Chimeric analogs should have the desirable effects of the parenthormones acting on their receptors, and therefore similar to the effectsof OXM, which apparently acts on both GLP-1R and GLCR: glucose-dependentinsulin secretion and satiety, coupled with lipolysis and increasedburning of fat due to glucagon. The analogs were shown to cause thedesired effects of decreased weight and increased burning of fat. Such aprofile would be attractive in the treatment of obesity, but a majorchallenge in obesity treatment is compliance. Although currently knownfull length analogs of glucagon and OXM, respectively, with affinity forboth GLP-1R and GLCR can result in weight loss, these analogs are notoptimized for the high bioavailability, pharmaceutical properties, andconvenient delivery to patients that are necessary for optimal drugtreatment regimens. Accordingly, provided herein are analogs of gutpeptides (e.g., GLP, OXM, glucagon or the like) that allow for highbioavailability and/or long lasting effects for improved therapeuticoutcome in treatment of conditions such as obesity and/or diabetesand/or the metabolic syndrome.

Additional factors for optimized treatment of the metabolic syndrome anddiabetes with OXM-like molecules relate to the duration of treatment andthe amount of glucagon action. For example, continuous treatment withanalogs that activate GLP-1 and glucagon receptors (the OXMpharmacological profile) can result in very large and rapid loss of fatmass (Day, J. W., et al. (2009) Nat Chem Biol 5: 749-757), but it canalso cause the loss of lean muscle mass (Kosinski, J. R., et al. (2012)Obesity (Silver Spring): doi: 10.1038/oby.2012.67), which is unfavorablefor a pharmaceutical in this class. For example, in the research articleby Kosinski, J. R., et al., the natural hormone Oxm is administeredcontinuously for 14 days from an Alzet minipump and results in adecrease of 30% in fat mass, but also caused a 7% decrease in lean mass(muscle).

Glucagon action is known to stimulate glycogenolysis, lipolysis and theincreased burning of fat, but can also have catabolic effects on muscle.A successful treatment using an agent that combines GLP-1 and glucagonaction (the OXM profile) will need to optimally cause the satiety andpotentiated glucose-dependent insulin secretion of a GLP-1 analog with ajudicious amount of glucagon action (fat burning). In addition,intermittent use of such an agent will provide the desired clinicalprofile of moderate, continuous weight loss, through loss of fat mass,with minimized loss of lean mass. Provided herein are molecules with adesirable combination of GLP-1 and OXM action as well as a tunablepharmacokinetic/pharmacodynamic profile to allow optimum use in therapy(for example in the metabolic syndrome, diabetes, obesity, and thelike).

In one embodiment, the compounds of Formula 3-I-A, 3-III-A, 3-III-B and3-V are designed to provide either glucagon-like activity or GLP-1 likeactivity. In a further embodiment, the compounds of Formula 3-I-A,3-III-A, 3-III-B and 3-V provide tunable activity. For example, in oneinstance, the peptide products described herein (e.g., compounds inTable 3 of FIG. 8 and Table 4 of FIG. 9) have an EC50 of less than about500 nM, preferably less than about 50 nM, more preferably less thanabout 20 nM at receptors for both glucagon, and GLP-1. In anotherinstance, the peptide products described herein (e.g., compounds inTable 3 of FIG. 8 and Table 4 of FIG. 9) are more potent (e.g., EC50 ofless than 10 nM, preferably less than 5 nM, more preferably about 1 nM)for the GLP-1 receptor and less potent for the glucagon receptor (e.g.,EC50 of less than 50 nM, preferably less than about 20 nM, morepreferably about 5 nM) for the glucagon receptor. This tunability ofbiological activity allows for some retention of a judicious amount ofglucagon action, thereby allowing for fat burning to occur, while alsoretaining the beneficial effects of potentiated glucose-dependentinsulin secretion. OXM is structurally homologous with GLP-1 andglucagon, and activates both the glucagon receptor (GCGR) and the GLP-1receptor (GLP1R). Accordingly, in some embodiments, the compounds ofFormula 3-I-A, 3-III-A, 3-III-B and 3-V provide a tunable OXM-likebiological activity. In some specific embodiments, the peptide productsdescribed herein comprise a peptide having amino acid residues 1-17 ofGLP-1 and/or analogs thereof (e.g., analogs comprising modifiednon-natural amino acid replacements as described herein, cyclized lactamlinkages as described herein, surfactant modifications as describedherein, or a combination thereof). In some other embodiments, thepeptide products described herein comprise a peptide having amino acidresidues 1-16 of GLP-1 and/or analogs thereof (e.g., analogs comprisingmodified non-natural amino acid replacements as described herein,cyclized lactam linkages as described herein, surfactant modificationsas described herein, or a combination thereof). In additionalembodiments, the peptide products described herein comprise a peptidehaving amino acid residues 1-18 of GLP-1 and/or analogs thereof (e.g.,analogs comprising modified non-natural amino acid replacements asdescribed herein, cyclized lactam linkages as described herein,surfactant modifications as described herein, or a combination thereof).Additionally the peptide products described herein comprise one or moreresidues (e.g., Aib, Ac4C) which provide helix stabilization of thedesigned compounds of Formula 3-I-A, 3-III-A, 3-III-B and 3-V, andcompounds in Table 3 of FIG. 8 and Table 4 of FIG. 9.

It is believed that the glucagon subfamily of ligands bind to theirreceptors in a two domain mode common to a number of the class B ofreceptors (secretin class, G Protein-coupled Receptors (GPCR)). ForGLP-1 it is felt that there is a N-terminal region of from residue 1 toabout residue 16 which binds to the tops of the transmembrane helicies(juxtomembrane region) and a helical C-terminal region from 17 to 31which binds to the large, extracellular, N-terminal extension (ECD) ofthe receptor. The binding of these ligands focuses on the fact thatN-terminally truncated analogs of these peptide ligands can still retainsubstantial binding affinity and selectivity for just the isolated ECDregion of the receptor. Therefore it has been suggested that theN-terminal region is responsible for receptor activation while theC-terminal region is responsible for binding. It recently has been shownthat short, N-terminal analogs of GLP-1 can be both potent binders aswell as receptor activators (Mapelli, C., et al. (2009) J Med Chem 52:7788-7799; Haque, T. S., et al. (2010) Peptides 31: 950-955; Haque, T.S., et al. (2010) Peptides 31: 1353-1360).

In addition, study of an x-ray crystal structure (Runge, S., et al.(2008) J Biol Chem 283: 11340-7) of the N-terminal region of the GLP1Rwith a truncated antagonist analogs of the GLP-1 mimic, exendin-4(Byetta), bound in this region show that a critical ligand-bindingregion in the ECD is of high hydrophobicity (FIG. 10). The sequence ofexendin-4 beyond Glu15 interacts as an amphiphilic helix with this veryhydrophobic region (Val^(19*), Phe^(22*), Trp^(25*), Leu^(26*)). In oneembodiment, truncated N-terminal fragments of GLP-1 or glucagon aremodified to bind to GLCR and are covalently linked to a surfactant. Thehydrophobic 1′-alkyl portion of the surfactant mimics and replaces theC-terminal region of the native hormone ligand and increases thepeptides potency, efficacy, and duration of action. In addition, suchanalogs have major advantages due to their smaller size, which reducestheir complexity, synthesis costs, and susceptibility to proteolysis. Inaddition smaller peptides are more readily absorbed through the nasalmucosa or gut enterocyte barrier.

Hypoglycemia is a condition of low blood sugar that can belife-threatening and is increasingly seen as more aggressive treatmentof elevated blood sugar by intensive insulin treatment is being used inmore patients. Hypoglycemia is seen when blood glucose levels drop toolow to provide enough energy to the brain and muscles for the body'sactivities. Glucagon can be used to treat this condition and does so bystimulating the liver to break down glycogen to generate glucose andcause the blood glucose levels to rise toward the normal value. Analogsof glucagon that retain the ability to activate the GLCR may be used toachieve this desirable effect on blood glucose levels.

Analogs of GLP-1 that activate the GLP1R stimulate the production and,in the presence of elevated blood glucose levels, release of insulinfrom the pancreas. This action results in efficient control andnormalization of blood glucose levels, as seen with current productssuch as exenatide (Byetta®). In addition, such products appear toproduce a decreased appetite and slow the movement of food from thestomach. Thus they are effective in treatment of diabetes throughmultiple mechanisms. Analogs that combine the effects of glucagon andGLP-1 that activate both the GLCR and the GLP1R may offer a benefit inthe treatment of diabetes through a concerted action to suppressappetite, release insulin in a glucose-dependent fashion, assist in theprotection from hypoglycemia and accelerate the burning of fat.

Such methods for treating hyperglycemia, including diabetes, diabetesmellitus type I, diabetes mellitus type II, or gestational diabetes,either insulin-dependent or non-insulin dependent, are expected to beuseful in reducing complications of diabetes including nephropathy,retinopathy and vascular disease. Applications in cardiovascular diseaseencompass microvascular as well as macrovascular disease (Davidson, M.H., (2011) Am J Cardiol 108[suppl]:33B-41B; Gejl, M., et al. (2012) JClin Endocrinol Metab 97:doi:10.1210/jc.2011-3456), and includetreatment for myocardial infarction. Such methods for reducing appetiteor promoting loss of body weight are expected to be useful in reducingbody weight, preventing weight gain, or treating obesity of variouscauses, including drug-induced obesity, and reducing complicationsassociated with obesity including vascular disease (coronary arterydisease, stroke, peripheral vascular disease, ischemia reperfusion,etc.), hypertension, onset of diabetes type II, hyperlipidemia andmusculoskeletal diseases.

As used herein, the term glucagon or GLP-1 analogs includes allpharmaceutically acceptable salts or esters thereof.

In one aspect, the peptides that are covalently modified and aresuitable for methods described herein are truncated analogs of glucagonand/or the related hormone GLP-1, including and not limited to:

Glucagon:

(SEQ. ID. NO. 632) His₁-Ser₂-Gln₃-Gly₄-Thr₅ Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Ser₁₆-Arg₁₇-Arg₁₈-Ala₁₉-Gln₂₀-Asp₂₁-Phe₂₂-Val₂₃-Gln₂₄-Trp₂₅- Leu₂₆-Met₂₇-Asn₂₈-Thr₂₉

Oxyntomodulin:

(SEQ. ID. NO. 633) His₁-Ser₂-Gln₃-Gly₄-Thr₅ Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Ser₁₆-Arg₁₇-Arg₁₈-Ala₁₉-Gln₂₀-Asp₂₁-Phe₂₂-Val₂₃-Gln₂₄-Trp₂₅-Leu₂₆-Met₂₇-Asn₂₈-Thr₂₉-Lys₃₀-Arg₃₁-Asn₃₂-Arg₃₃- Asn₃₄-Asn₃₅-Ile₃₆-Ala₃₇

GLP-1 (Using Glucagon Numbering):

(SEQ. ID. NO. 634) His₁-Ala₂-Glu₃-Gly₄-Thr₅ Phe₆-Thr₇-Ser₈-Asp₉-Val₁₀-Ser₁₁-Ser₁₂-Tyr₁₃-Leu₁₄-Glu₁₅-Gly₁₆-Gln₁₇-Ala₁₈-Ala₁₉-Lys₂₀-Glu₂₁-Phe₂₂-Ile₂₃-Ala₂₄-Trp₂₅-Leu₂₆-Val₂₇-Lys₂₈-Gly₂₉-Arg₃₀

In some embodiments, a peptide product described herein has thestructure of Formula 3-V:

FORMULA 3-V (SEQ. ID. NO. 635)aa₁-aa₂-aa₃-aa₄-aa₅-aa₆-aa₇-aa₈-aa₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇-aa₁₈-aa₁₉-aa₂₀-aa₂₁-aa₂₂-aa₂₃-aa₂₄-aa₂₅-aa₂₆-aa₂₇-aa₂₈-aa₂₉-aa₃₀-aa₃₁-aa₃₂-aa₃₃-aa₃₄-aa₃₅-aa₃₆-aa₃₇-Zwherein:

-   -   U is a linking amino acid;    -   X is a surfactant-linked to the side chain of U;    -   Z is OH, or —NH—R³, wherein R³ is H or C₁-C₁₂ substituted or        unsubstituted alklyl;    -   aa₁ is His, N—Ac-His, pGlu-His or N—R³-His;    -   aa₂ is Ser, Ala, Gly, Aib, Ac4c or Ac5c;    -   aa₃ is Gln, or Cit;    -   aa₄ is Gly, or D-Ala;    -   aa₅ is Thr, or Ser;    -   aa₆ is Phe, Trp, F2Phe, Me2Phe, or Nal(2);    -   aa₇ is Thr, or Ser;    -   aa₈ is Ser, or Asp;    -   aa₉ is Asp, or Glu;    -   aa₁₀ is Tyr, Leu, Met, Nal(2), Bip, or Bip2EtMeO;    -   aa₁₁ is Ser, Asn, or U(X);    -   aa₁₂ is Lys, Glu, Ser, Arg, or U(X);    -   aa₁₃ is absent, Tyr, Gln, Cit, or U(X);    -   aa₁₄ is absent, Leu, Met, Nle, or U(X);    -   aa₁₅ is absent, Asp, Glu, or U(X);    -   aa₁₆ is absent, Ser, Gly, Glu, Aib, Ac5c, Lys, Arg, or U(X);    -   aa₁₇ is absent, Arg, hArg, Gln, Glu, Cit, Aib, Ac4c, Ac5c, or        U(X);    -   aa₁₈ is absent, Arg, hArg, Ala, Aib, Ac4c, Ac5c, or U(X);    -   aa₁₉ is absent, Ala, Val, Aib, Ac4c, Ac5c, or U(X);    -   aa₂₀ is absent, Gln, Lys, Arg, Cit, Glu, Aib, Ac4c, Ac5c, or        U(X);    -   aa₂₁ is absent, Asp, Glu, Leu, Aib, Ac4c, Ac5c, or U(X);    -   aa₂₂ is absent, Phe, Trp, Nal(2), Aib, Ac4c, Ac5c, or U(X);    -   aa₂₃ is absent, Val, Ile, Aib, Ac4c, Ac5c, or U(X);    -   aa₂₄ is absent, Gln, Ala, Glu, Cit, or U(X);    -   aa₂₅ is absent, Trp, Nal(2), or U(X);    -   aa₂₆ is absent, Leu, U(X);    -   aa₂₇ is absent, Met, Val, Nle, Lys, or U(X);    -   aa₂₈ is absent, Asn, Lys, or U(X);    -   aa₂₉ is absent, Thr, Gly, Aib, Ac4c, Ac5c, or U(X);    -   aa₃₀ is absent, Lys, Aib, Ac4c, Ac5c, or U(X);    -   aa₃₁ is absent, Arg, Aib, Ac4c, Ac5c, or U(X);    -   aa₃₂ is absent, Asn, Aib, Ac4c, Ac5c, or U(X);    -   aa₃₃ is absent, Arg, Aib, Ac5c, or U(X);    -   aa₃₄ is absent, Asn, Aib, Ac4c, Ac5c, or U(X);    -   aa₃₅ is absent, Asn, Aib, Ac4c, Ac5c, or U(X);    -   aa₃₆ is absent, Ile, Aib, Ac4c, Ac5C, or U(X);    -   aa₃₆ is absent, Ala, Aib, Ac4c, Ac5C, or U(X);    -   aa₃₇ absent or U(X);

provided that one, or at least one of aa₁₁-aa₃₇ is U(X).

In specific embodiments, the linking amino acid U, is a diamino acidlike Lys or Orn, X is a modified surfactant from the 1-alkyl glycosideclass linked to U, and Z is OH, or —NH—R₂, wherein R³ is H or C₁-C₁₂; ora PEG chain of less than 10 Da.

In some embodiments, a peptide product described herein has thestructure of Formula III-B:

FORMULA 3-III-B (SEQ. ID. NO. 305)His₁-aa₂-aa₃-Gly₄-Thr₅-aa₆-Thr₇-Ser₈-Asp₉-aa₁₀-aa₁₁-aa₁₂-aa₁₃-aa₁₄-aa₁₅-aa₁₆-aa₁₇-aa₁₈-aa₁₉-aa₂₀- aa₂₁-aa₂₂-aa₂₃-Zwherein:

-   -   Z is OH, or —NH—R³, wherein R³ is H or substituted or        unsubstituted C₁-C₁₂ alkyl; or a PEG chain of less than 10 Da;        -   aa₂ is Ser, Ala, Gly, Aib, Ac4c, or Ac5c;        -   aa₃ is Gln, or Cit;        -   aa₆ is Phe, Trp, F2Phe, Me2Phe, MePhe, or Nal2;        -   aa₁₀ is Tyr, Leu, Met, Nal2, Bip, or Bip2EtMeO;        -   aa₁₁ is Ser, Asn, or U;        -   aa₁₂ is Lys, Glu, Ser or U(X);        -   aa₁₃ is absent or Tyr, Gln, Cit, or U(X);        -   aa₁₄ is absent or Leu, Met, Nle, or U(X);        -   aa₁₅ is absent or Asp, Glu, or U(X);        -   aa₁₆ is absent or Ser, Gly, Glu, Aib, Ac4c, Ac5c, Lys, R, or            U(X);        -   aa₁₇ is absent or Arg, hArg, Gln, Glu, Cit, Aib, Ac4c, Ac5c,            or U(X);        -   aa₁₈ is absent or Arg, hArg, Ala, Aib, Ac4c, Ac5c, or U(X);        -   aa₁₉ is absent or Ala, Val, Aib, Ac4c, Ac5c, or U(X);        -   aa₂₀ is absent or Gln, Lys, Arg, Cit, Glu, Aib, Ac4c, Ac5c,            or U(X);        -   aa₂₁ is absent or Asp, Glu, Leu, Aib, Ac4c, Ac5c, or U(X);        -   aa₂₂ is absent or Phe, Aib, Ac4c, Ac5c, or U(X)        -   aa₂₃ is absent or Val, Ile, Aib, Ac4c, Ac5c, or U(X);    -   wherein any two of aa₁-aa₂₃ are optionally cyclized through        their side chains to form a lactam linkage; and    -   provided that one, or at least one of aa₁₆, aa₁₇, aa₁₈, aa₁₉,        aa₂₀, aa₂₁, aa₂₂, aa₂₃ or aa₂₄ is the natural or unnatural amino        acid U covalently attached to X.

In some specific embodiments of Formula 3-III-A, Formula 3-III-B andFormula 3-V, X has the structure:

-   -   wherein:        -   R^(1a) is a substituted or unsubstituted C₁-C₃₀ alkyl group;        -   R^(1b), R^(1c), and R^(1d) are H;        -   W¹ is —(C═O)—NH—;        -   W² is —O—; and        -   R² is a bond.

In some of the embodiments described above, R^(1a) is a C₁-C₂₀ alkylgroup, a C₈-C₂₀ alkyl group, C₁₂-18 alkyl group or C₁₄-C₁₈ alkyl group.

In some embodiments of Formula 3-III-B, U is any linker amino aciddescribed herein. Table 3 of FIG. 8 and Table 4 of FIG. 9 illustratecertain examples of peptides that covalently linked with surfactants asdescribed herein.

Contemplated within the scope of embodiments presented herein arepeptide products of Formula 3-I-A, Formula 3-III-A, Formula 3-III-B orFormula 3-V, wherein the peptide product comprises one, or, more thanone surfactant groups (e.g., group X having the structure of Formula3-I). In one embodiment, a peptide product of Formula 3-I-A, Formula3-III-A, Formula 3-III-B or Formula 3-V, comprises one surfactant group.In another embodiment, a peptide product of Formula 3-I-A, Formula3-III-A, Formula 3-III-B or Formula 3-V, comprises two surfactantgroups. In yet another embodiment, a peptide product of Formula 3-I-A,Formula 3-III-A, Formula 3-III-B or Formula 3-V, comprises threesurfactant groups.

Recognized herein is the importance of certain portions of SEQ. ID. NO.632 for the treatment of conditions associated with insulin resistanceand/or cardiovascular conditions. Accordingly, provided herein is amethod of treating diabetes in an individual in need thereof comprisingadministration of a therapeutically effective amount of a glucagonanalog comprising amino acid residues aa₁-aa₁₇ of SEQ. ID. NO. 632 tothe individual in need thereof.

In a further embodiment, provided herein is a method of treatingdiabetes in an individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₁₈ of SEQ. ID. NO. 632 to the individual in needthereof.

In another embodiment, provided herein is a method of treating diabetesin an individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₁₉ of SEQ. ID. NO. 632 to the individual in needthereof.

In another embodiment, provided herein is a method of treating diabetesin an individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₂₀ of SEQ. ID. NO. 632 to the individual in needthereof.

In an additional embodiment, the administration of the said glucagonanalog described above causes weight loss.

Recognized herein is the importance of certain portions of SEQ. ID. NO.303 for the treatment of conditions associated with insulin resistanceand/or cardiovascular conditions. Accordingly, provided herein is amethod of treating diabetes in an individual in need thereof comprisingadministration of a therapeutically effective amount of a glucagonanalog comprising amino acid residues aa₁-aa₁₇ of SEQ. ID. NO. 303 tothe individual in need thereof.

In a further embodiment, provided herein is a method of treatingdiabetes in an individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₁₈ of SEQ. ID. NO. 303 to the individual in needthereof.

In another embodiment, provided herein is a method of treating diabetesin an individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₁₉ of SEQ. ID. NO. 303 to the individual in needthereof.

In another embodiment, provided herein is a method of treating diabetesin an individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₂₀ of SEQ. ID. NO. 303 to the individual in needthereof.

In an additional embodiment, the administration of the said glucagonanalog described above causes weight loss.

In any of the embodiments described above, the said glucagon analog ismodified with a surfactant X of Formula 3-I:

-   -   wherein:        -   R^(1a) is independently, at each occurrence, a bond, H, a            substituted or unsubstituted C₁-C₃₀ alkyl group, a            substituted or unsubstituted alkoxyaryl group, a substituted            or unsubstituted aralkyl group, or a steroid nucleus            containing moiety;        -   R^(1b), R^(1c), and R^(1d) are each, independently at each            occurrence, a bond, H, a substituted or unsubstituted C₁-C₃₀            alkyl group, a substituted or unsubstituted alkoxyaryl            group, or a substituted or unsubstituted aralkyl group;        -   W¹ is independently, at each occurrence, —CH₂—, —CH₂—O—,            —(C═O), —(C═O)—O—, —(C═O)—NH—, —(C═S)—, —(C═S)—NH—, or            —CH₂—S—;        -   W² is —O—, —CH₂— or —S—;        -   R² is independently, at each occurrence, a bond to U, H, a            substituted or unsubstituted C₁-C₃₀ alkyl group, a            substituted or unsubstituted alkoxyaryl group, or a            substituted or unsubstituted aralkyl group, —NH₂, —SH,            C₂-C₄-alkene, C₂-C₄-alkyne, —NH(C═O)—CH₂—Br,            —(CH₂)_(m)-maleimide, or —N₃;        -   n is 1, 2 or 3; and        -   m is 1-10.

In a specific embodiment, the said glucagon analog is modified with asurfactant, X having the structure:

-   -   wherein:        -   R^(1a) is a substituted or unsubstituted C₁-C₃₀ alkyl group;        -   R^(1b), R^(1c), and R^(1d) are H;        -   W¹ is —(C═O)—NH—;        -   W² is —O—; and        -   R² is a bond.

In some of the embodiments described above, R^(1a) is a C₁-C₂₀ alkylgroup, a C₈-C₂₀ alkyl group, C₁₂-C₁₈ alkyl group or C₁₄-C₁₈ alkyl group.

Also provided herein is a method of treating a cardiovascular disesasein an individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₁₇ of SEQ. ID. NO. 632 to the individual in needthereof.

Also provided herein is a method of treating a cardiovascular disesasein an individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₁₈ of SEQ. ID. NO. 632 to the individual in needthereof.

Also provided herein is a method of treating a cardiovascular disesasein an individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₁₉ of SEQ. ID. NO. 632 to the individual in needthereof.

Also provided herein is a method of treating a cardiovascular disesasein an individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₂₀ of SEQ. ID. NO. 632 to the individual in needthereof.

In some cases for the embodiments described abvoe, the said glucagonanalog is administered when the cardiovascular disease is associatedwith an ischemic event.

Also provided herein is a method of treating a cardiovascular disesasein an individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₁₇ of SEQ. ID. NO. 303 to the individual in needthereof.

Also provided herein is a method of treating a cardiovascular disesasein an individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₁₈ of SEQ. ID. NO. 303 to the individual in needthereof.

Also provided herein is a method of treating a cardiovascular disesasein an individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₁₉ of SEQ. ID. NO. 303 to the individual in needthereof.

Also provided herein is a method of treating a cardiovascular disesasein an individual in need thereof comprising administration of atherapeutically effective amount of a glucagon analog comprising aminoacid residues aa₁-aa₂₀ of SEQ. ID. NO. 303 to the individual in needthereof.

In some cases for the embodiments described abvoe, the said glucagonanalog is administered when the cardiovascular disease is associatedwith an ischemic event.

In any of the embodiments described above, the said glucagon analog ismodified with a surfactant X of Formula 3-I:

-   -   wherein:        -   R^(1a) is independently, at each occurrence, a bond, H, a            substituted or unsubstituted C₁-C₃₀ alkyl group, a            substituted or unsubstituted alkoxyaryl group, a substituted            or unsubstituted aralkyl group, or a steroid nucleus            containing moiety;        -   R^(1b), R^(1c), and R^(1d) are each, independently at each            occurrence, a bond, H, a substituted or unsubstituted C₁-C₃₀            alkyl group, a substituted or unsubstituted alkoxyaryl            group, or a substituted or unsubstituted aralkyl group;        -   W¹ is independently, at each occurrence, —CH₂—, —CH₂—O—,            —(C═O), —(C═O)—O—, —(C═O)—NH—, —(C═S)—, —(C═S)—NH—, or            —CH₂—S—;        -   W² is —O—, —CH₂— or —S—;        -   R² is independently, at each occurrence, a bond to U, H, a            substituted or unsubstituted C₁-C₃₀ alkyl group, a            substituted or unsubstituted alkoxyaryl group, or a            substituted or unsubstituted aralkyl group, —NH₂, —SH,            C₂-C₄-alkene, C₂-C₄-alkyne, —NH(C═O)—CH₂—Br,            —(CH₂)_(m)-maleimide, or —N₃;        -   n is 1, 2 or 3; and        -   m is 1-10.

In a specific embodiment, the said glucagon analog is modified with asurfactant, X having the structure:

-   -   wherein:        -   R^(1a) is a substituted or unsubstituted C₁-C₃₀ alkyl group;        -   R^(1b), R^(1c), and R^(1d) are H;        -   W¹ is —(C═O)—NH—;        -   W² is —O—; and        -   R² is a bond.

In some of the embodiments described above, R^(1a) is a C₁-C₂₀ alkylgroup, a C₈-C₂₀ alkyl group, C₁₂-C₁₈ alkyl group or C₁₄-C₁₈ alkyl group.

Modifications at the amino or carboxyl terminus may optionally beintroduced into the peptides (e.g., glucagon or GLP-1) (Nestor, J. J.,Jr. (2009) Current Medicinal Chemistry 16: 4399-4418). For example, thepeptides can be truncated or acylated on the N-terminus to yieldpeptides analogs exhibiting low efficacy, partial agonist and antagonistactivity, as has been seen for some peptides (Gourlet, P., et al. (1998)Eur J Pharmacol 354: 105-111, Gozes, I. and Furman, S. (2003) Curr PharmDes 9: 483-494), the contents of which is incorporated herein byreference). For example, deletion of the first 6 residues of bPTH yieldsantagonistic analogs (Mahaffey, J. E., et al. (1979) J Biol Chem 254:6496-6498; Goldman, M. E., et al. (1988) Endocrinology 123: 2597-2599)and a similar operation on peptides described herein generates potentantagonistic analogs. Other modifications to the N-terminus of peptides,such as deletions or incorporation of D-amino acids such as D-Phe alsocan give potent and long acting agonists or antagonists when substitutedwith the modifications described herein such as long chain alkylglycosides. Such agonists and antagonists also have commercial utilityand are within the scope of contemplated embodiments described herein.

Also contemplated within the scope of embodiments described herein aresurfactants covalently attached to peptide analogs, wherein the nativepeptide is modified by acetylation, acylation, PEGylation,ADP-ribosylation, amidation, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-link formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, (Nestor, J. J., Jr. (2007) Comprehensive Medicinal ChemistryII 2: 573-601, Nestor, J. J., Jr. (2009) Current Medicinal Chemistry 16:4399-4418, Creighton, T. E. (1993, Wold, F. (1983) PosttranslationalCovalent Modification of Proteins 1-12, Seifter, S. and Englard, S.(1990) Methods Enzymol 182: 626-646, Rattan, S. I., et al. (1992) Ann NY Acad Sci 663: 48-62). Also contemplated within the scope ofembodiments described herein are peptides that are branched or cyclic,with or without branching. Cyclic, branched and branched circularpeptides result from post-translational natural processes and are alsomade by suitable synthetic methods. In some embodiments, any peptideproduct described herein comprises a peptide analog described above thatis then covalently attached to an alkyl-glycoside surfactant moiety.

Also contemplated within the scope of embodiments presented herein arepeptide chains substituted in a suitable position by the substitution ofthe analogs claimed herein by acylation on a linker amino acid, at forexample the ε-position of Lys, with fatty acids such as octanoic,decanoic, dodecanoic, tetradecanoic, hexadecanoic, octadecanoic,3-phenylpropanoic acids and the like, with saturated or unsaturatedalkyl chains (Zhang, L. and Bulaj, G. (2012) Curr Med Chem 19:1602-1618). Non-limiting, illustrative examples of such analogs are:

(SEQ. ID. NO. 636) His₁-Aib₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Ser₁₆-Arg₁₇-Lys(N-epsilon-dodecanoyl)₁₈-Aib₁₉-NH₂, (SEQ. ID. NO. 637)His₁-Aib₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Ser₁₆-Arg₁₇-Lys(N-epsilon-tetradecanoyl)₁₈-Ac4c₁₉-NH₂, (SEQ. ID. NO. 638)His₁-Aib₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Ser₁₆-Arg₁₇-Lys(N-epsilon-hexadecanoyl)₁₈-Aib₁₉-NH₂, (SEQ. ID. NO. 639)His₁-Aib₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Aib₁₆-Arg₁₇-Lys(N-epsilon-dodecanoyl)₁₈-NH₂, (SEQ. ID. NO. 640)His₁-Aib₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Aib₁₆-Arg₁₇-Lys(N-epsilon-tetradecanoyl)₁₈-NH₂, (SEQ. ID. NO. 641)His₁-Aib₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Aib₁₆-Arg₁₇-Lys(N-epsilon-hexadecanoyl)₁₈-NH₂, (SEQ. ID. NO. 642)His₁-Aib₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Ser₁₆-Arg₁₇-Lys(N-epsilon-(gamma-glutamyl)-N-alpha- tetradecanoyl))₁₈-Aib₁₉-NH₂, andthe like.

In further embodiments, a peptide chain is optionally substituted in asuitable position by reaction on a linker amino acid, for example thesulfhydryl of Cys, with a spacer and a hydrophobic moiety such as asteroid nucleus, for example a cholesterol moiety. In some of suchembodiments, the modified peptide further comprises one or more PEGchains. Non-limiting examples of such molecules are:

His₁-Aib₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-Lys₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Aib₁₆-Arg₁₇-Cys(S-(3-(PEG4-aminoethylacetamide-Cholesterol)))₁₈-Aib₁₉-NH₂(SEQ. ID. NO. 643),His₁-Aib₂-Gln₃-Gly₄-Thr₅-Phe₆-Thr₇-Ser₈-Asp₉-Tyr₁₀-Ser₁₁-cyclo(Glu₁₂-Tyr₁₃-Leu₁₄-Asp₁₅-Lys₁₆)-Arg₁₇-Cys(S-(3-(PEG4-aminoethylacetamide-Cholesterol)))₁₈-NH₂(SEQ. ID. NO. 644).

Aside from the twenty standard amino acids, there are a vast number of“nonstandard amino acids” or unnatural amino acids that are known to theart and that may be incorporated in the compounds described herein, asdescribed above. Other nonstandard amino acids are modified withreactive side chains for conjugation (Gauthier, M. A. and Klok, H. A.(2008) Chem Commun (Camb) 2591-2611; de Graaf, A. J., et al. (2009)Bioconjug Chem 20: 1281-1295). In one approach, an evolved tRNA/tRNAsynthetase pair and is coded in the expression plasmid by the ambersuppressor codon (Deiters, A, et al. (2004). Bio-org. Med. Chem. Lett.14, 5743-5). For example, p-azidophenylalanine was incorporated intopeptides and then reacted with a functionalized surfactant, or a PEGpolymer having an acetylene moiety in the presence of a reducing agentand copper ions to facilitate an organic reaction known as “Huisgen[3+2] cycloaddition.” A similar reaction sequence using the reagentsdescribed herein containing an acetylene modified alkyl glycoside or PEGmodified glycoside will result in PEGylated or alkyl glycoside modifiedpeptides. For peptides of less than about 50 residues, standard solidphase synthesis is used for incorporation of said reactive amino acidresidues at the desired position in the chain. Such surfactant-modifiedpeptides and/or proteins offer a different spectrum of pharmacologicaland medicinal properties than peptides modified by PEG incorporationalone.

The skilled artisan will appreciate that numerous permutations of thepeptide analogs are possible and, provided that an amino acid sequencehas an incorporated surfactant moiety, will possess the desirableattributes of surfactant modified peptide products described herein.

Certain Definitions

As used in the specification, “a” or “an” means one or more. As used inthe claim(s), when used in conjunction with the word “comprising,” thewords “a” or “an” mean one or more. As used herein, “another” means atleast a second or more.

As used herein, the one- and three-letter abbreviations for the variouscommon amino acids are as recommended in Pure Appl. Chem. 31, 639-645(1972) and 40, 277-290 (1974) and comply with 37 CFR § 1.822 (55 FR18245, May 1, 1990). The abbreviations represent L-amino acids unlessotherwise designated as D- or DL. Certain amino acids, both natural andnon-natural, are achiral, e.g., glycine, α-amino-isobutyric acid (Aib).All peptide sequences are presented with the N-terminal amino acid onthe left and the C-terminal amino acid on the right.

An “alkyl” group refers to an aliphatic hydrocarbon group. Reference toan alkyl group includes “saturated alkyl” and/or “unsaturated alkyl”,i.e., an alkene or an alkyne. The alkyl group, whether saturated orunsaturated, includes branched, straight chain, or cyclic groups. Analkyl group is optionally substituted with substituents including andnot limited to oxo, halogen, aryl, cycloalkyl, hydrophobic naturalproduct such as a steroid, an aralkyl chain (including alkoxyaryl),alkyl chain containing an acyl moiety, or the like. In some embodiments,an alkyl group is linked to the Nα-position of a residue (e.g., Tyr orDmt) in a peptide. This class is referred to as N-alkyl and comprisesstraight or branched alkyl groups from C₁-C₁₀, or an aryl substitutedalkyl group such as benzyl, phenylethyl and the like. In someembodiments, an alkyl moiety is a 1-alkyl group that is in glycosidiclinkage (typically to the 1-position of, for example, glucose) to thesaccharide moiety. Such a 1-alkyl group is a C₁-C₃₀ alkyl group.

An “aryl” group refers to an aromatic ring wherein each of the atomsforming the ring is a carbon atom. Aryl rings described herein includerings having five, six, seven, eight, nine, or more than nine carbonatoms. Aryl groups are optionally substituted with substituents selectedfrom halogen, alkyl, acyl, alkoxy, alkylthio, sulfonyl, dialkyl-amino,carboxyl esters, cyano or the like. Examples of aryl groups include, butare not limited to phenyl, and naphthalenyl.

The term “acyl” refers to a C₁-C₂₀ acyl chain. This chain may comprise alinear aliphatic chain, a branched aliphatic chain, a chain containing acyclic alkyl moiety, a hydrophobic natural product such as a steroid, anaralkyl chain, or an alkyl chain containing an acyl moiety.

The term “steroid nucleus” refers to the core of steroids comprising anarrangement of four fused rings designated A, B, C and D as shown below:

Examples of steroid nucleus containing moieties include, and are notlimited to, cholesterol and the like.

As used herein, a “therapeutic composition” can comprise an admixturewith an aqueous or organic carrier or excipient, and can be compounded,for example, with the usual nontoxic, pharmaceutically acceptablecarriers for tablets, pellets, capsules, lyophilizates, suppositories,solutions, emulsions, suspensions, or other form suitable for use. Thecarriers, in addition to those disclosed above, can include alginate,collagen, glucose, lactose, mannose, gum acacia, gelatin, mannitol,starch paste, magnesium trisilicate, talc, corn starch, keratin,colloidal silica, potato starch, urea, medium chain lengthtriglycerides, dextrans, and other carriers suitable for use inmanufacturing preparations, in solid, semisolid, or liquid form. Inaddition, auxiliary stabilizing, thickening or coloring agents can beused, for example a stabilizing dry agent such as triulose.

As used herein, a “pharmaceutically acceptable carrier” or “therapeuticeffective carrier” is aqueous or nonaqueous (solid), for examplealcoholic or oleaginous, or a mixture thereof, and can contain asurfactant, emollient, lubricant, stabilizer, dye, perfume,preservative, acid or base for adjustment of pH, a solvent, emulsifier,gelling agent, moisturizer, stabilizer, wetting agent, time releaseagent, humectant, or other component commonly included in a particularform of pharmaceutical composition. Pharmaceutically acceptable carriersare well known in the art and include, for example, aqueous solutionssuch as water or physiologically buffered saline or other solvents orvehicles such as glycols, glycerol, and oils such as olive oil orinjectable organic esters. A pharmaceutically acceptable carrier cancontain physiologically acceptable compounds that act, for example, tostabilize or to increase the absorption of specific inhibitor, forexample, carbohydrates, such as glucose, sucrose or dextrans,antioxidants, such as ascorbic acid or glutathione, chelating agents,low molecular weight proteins or other stabilizers or excipients.

The pharmaceutical compositions can also contain other pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such “substances” include, but are not limited to, pHadjusting and buffering agents, tonicity adjusting agents and the like,for example, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, etc. Additionally, the peptide, or variantthereof, suspension may include lipid-protective agents which protectlipids against free-radical and lipid-peroxidative damages on storage.Lipophilic free-radical quenchers, such as alpha-tocopherol andwater-soluble iron-specific chelators, such as ferrioxamine, aresuitable.

As used herein, a “surfactant” is a surface active agent that modifiesinterfacial tension of water. Typically, surfactants have one lipophilicand one hydrophilic group or region in the molecule. Broadly, the groupincludes soaps, detergents, emulsifiers, dispersing and wetting agents,and several groups of antiseptics. More specifically, surfactantsinclude stearyltriethanolamine, sodium lauryl sulfate, sodiumtaurocholate, laurylaminopropionic acid, lecithin, benzalkoniumchloride, benzethonium chloride and glycerin monostearate; andhydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone,polyethyleneglycol (PEG), carboxymethylcellulose sodium,methylcellulose, hydroxymethylcellulose, hydroxyethyl cellulose andhydroxypropylcellulose or alkyl glycosides. In some embodiments, asurfactant is a non-ionic surfactant (e.g., an alkyl glycosidesurfactant). In some embodiments, a surfactant is an ionic surfactant.

As used herein, “alkyl glycoside” refers to any sugar joined by alinkage to any hydrophobic alkyl, as is known in the art. Thehydrophobic alkyl can be chosen of any desired size, depending on thehydrophobicity desired and the hydrophilicity of the saccharide moiety.In one aspect, the range of alkyl chains is from 1 to 30 carbon atoms;or from 6 to 16 carbon atoms.

As used herein, “saccharide” is inclusive of monosaccharides,oligosaccharides or polysaccharides in straight chain or ring forms.Oligosaccharides are saccharides having two or more monosaccharideresidues. Some examples of the many possible saccharides suitable foruse in functionalized form include glucose, galactose, maltose,melibiose, maltotriose, maltotetraose, sucrose, trehalose or the like.

As used herein, “sucrose esters” are sucrose esters of fatty acids.Sucrose esters can take many forms because of the eight hydroxyl groupsin sucrose available for reaction and the many fatty acid groups, fromacetate on up to larger, more bulky fats that can be reacted withsucrose. This flexibility means that many products and functionalitiescan be tailored, based on the fatty acid moiety used. Sucrose estershave food and non-food uses, especially as surfactants and emulsifiers,with growing applications in pharmaceuticals, cosmetics, detergents andfood additives. They are biodegradable, non-toxic and mild to the skin.

As used herein, a “suitable” alkyl glycoside means one that is nontoxicand nonionic. In some instances, a suitable alkyl glycoside reduces theimmunogenicity or aggregation and increases the bioavailability of acompound when it is administered with the compound via the ocular,nasal, nasolacrimal, sublingual, buccal, inhalation routes or byinjection routes such as the subcutaneous, intramuscular, or intravenousroutes. Suitable compounds can be determined using the methods known tothe art and those set forth in the examples.

A “linker amino acid” is any natural or unnatural amino acid thatcomprises a reactive functional group (de Graaf, A. J., et al. (2009)Bioconjug Chem 20: 1281-1295) that is used for covalent linkage with thefunctionalized surfactant. By way of example, in some embodiments, alinker amino acid is Lys, or Orn having a reactive functional group—NH₂; or Cys, having a reactive functional group —SH; or Asp or Glu,having a reactive functional group —C(═O)—OH. By way of example, in someother embodiments, a linker amino acid is any amino acid having areactive functional group such as —OH, —N₃, haloacetyl or an acetylenicgroup that is used for formation of a covalent linkage with a suitablyfunctionalized surfactant.

As used herein, a “functionalized surfactant” is a surfactant comprisinga reactive group suitable for covalent linkage with a linker amino acid.By way of example, in some embodiments, a functionalized surfactantcomprises a carboxylic acid group (e.g., at the 6-position of amonosaccharide) as the reactive group suitable for covalent linkage witha linker amino acid. By way of example, in some embodiments, afunctionalized surfactant comprises a —NH₂ group, a —N₃ group, anacetylenic group, a haloacetyl group, a —O—NH₂ group, or a—(CH₂-)m-maleimide group, e.g., at the 6-position of a monosaccharide(as shown in Scheme 6), that allows for covalent linkage with a suitablelinker amino acid. In some embodiments, a functionalized surfactant is acompound of Formula IV as described herein.

As used herein, the term “peptide” is any peptide comprising two or moreamino acids. The term peptide includes short peptides (e.g., peptidescomprising between 2-14 amino acids), medium length peptides (15-50) orlong chain peptides (e.g., proteins). The terms peptide, medium lengthpeptide and protein may be used interchangeably herein. As used herein,the term “peptide” is interpreted to mean a polymer composed of aminoacid residues, related naturally occurring structural variants, andsynthetic non-naturally occurring analogs thereof linked via peptidebonds, related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic peptides can besynthesized, for example, using an automated peptide synthesizer.

Peptides may contain amino acids other than the 20 gene encoded aminoacids. “Peptide(s)” include those modified either by natural processes,such as processing and other post-translational modifications, but alsoby chemical modification techniques. Such modifications are welldescribed in basic texts and in more detailed monographs, and arewell-known to those of skill in the art. It will be appreciated that insome embodiments, the same type of modification is present in the sameor varying degree at several sites in a given peptide. Also, a givenpeptide, in some embodiments, contains more than one type ofmodifications. Modifications occur anywhere in a peptide, including thepeptide backbone, the amino acid side-chains, and the amino or carboxyltermini.

Accordingly, also contemplated within the scope of embodiments describedherein are surfactants covalently attached to peptides that aremodified, including, for example, modification by, acetylation,acylation, PEGylation, ADP-ribosylation, amidation, covalent attachmentof a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-link formation ofcysteine, formation of pyroglutamate, formylation, gamma-carboxylation,glycosylation, GPI anchor formation, hydroxylation, iodination,methylation, myristoylation, oxidation, proteolytic processing,phosphorylation, prenylation, racemization, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins, such asarginylation, and ubiquitination. See, for instance, (Nestor, J. J., Jr.(2007) Comprehensive Medicinal Chemistry II 2: 573-601, Nestor, J. J.,Jr. (2009) Current Medicinal Chemistry 16: 4399-4418, Creighton, T. E.(1993, Wold, F. (1983) Posttranslational Covalent Modification ofProteins 1-12, Seifter, S. and Englard, S. (1990) Methods Enzymol 182:626-646, Rattan, S. I., et al. (1992) Ann N Y Acad Sci 663: 48-62). Alsocontemplated within the scope of embodiments described herein arepeptides that are branched or cyclic, with or without branching. Cyclic,branched and branched circular peptides result from post-translationalnatural processes and are also made by suitable synthetic methods.

The term peptide includes peptides or proteins that comprise natural andunnatural amino acids or analogs of natural amino acids. As used herein,peptide and/or protein “analogs” comprise non-natural amino acids basedon natural amino acids, such as tyrosine analogs, which includespara-substituted tyrosines, ortho-substituted tyrosines, and metasubstituted tyrosines, wherein the substituent on the tyrosine comprisesan acetyl group, a benzoyl group, an amino group, a hydrazine, anhydroxyamine, a thiol group, a carboxy group, a methyl group, anisopropyl group, a C₂-C₂₀ straight chain or branched hydrocarbon, asaturated or unsaturated hydrocarbon, an O-methyl group, a polyethergroup, a halogen, a nitro group, or the like. Examples of Tyr analogsinclude 2,4-dimethyl-tyrosine (Dmt), 2,4-diethyl-tyrosine,O-4-allyl-tyrosine, 4-propyl-tyrosine, Ca-methyl-tyrosine and the like.Examples of lysine analogs include ornithine (Orn), homo-lysine,Ca-methyl-lysine (CMeLys), and the like. Examples of phenylalanineanalogs include, but are not limited to, meta-substitutedphenylalanines, wherein the substituent comprises a methoxy group, aC₁-C₂₀ alkyl group, for example a methyl group, an allyl group, anacetyl group, or the like. Specific examples include, but are notlimited to, 2,4,6-trimethyl-L-phenylalanine (Tmp), O-methyl-tyrosine,3-(2-naphthyl)alanine (Nal(2)), 3-(1-naphthyl)alanine (Nal(1)),3-methyl-phenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic), fluorinated phenylalanines, isopropyl-phenylalanine,p-azido-phenylalanine, p-acyl-phenylalanine, p-benzoyl-phenylalanine,p-iodo-phenylalanine, p-bromophenylalanine, p-amino-phenylalanine, andisopropyl-phenylalanine, and the like. Among the vast array ofnonstandard or unnatural amino acids known to the art and used inpeptide analog design are C-alpha-disubstituted amino acids such as Aib,Ca-diethylglycine (Deg), aminocyclopentane-1-carboxylic acid (Ac4c),aminocyclopentane-1-carboxylic acid (Ac5c), and the like. Such aminoacids frequently lead to a restrained structure, often biased toward analpha helical structure (Kaul, R. and Balaram, P. (1999) Bioorg Med Chem7: 105-117). Additional examples of such unnatural amino acids useful inanalog design are homo-arginine (Har), and the like. Substitution ofreduced amide bonds in certain instances leads to improved protectionfrom enzymatic destruction or alters receptor binding. By way ofexample, incorporation of a Tic-Phe dipeptide unit with a reduced amidebond between the residues (designated as Tic-Ψ[CH2-NH]-Ψ-Phe) reducesenzymatic degradation. Accordingly, also contemplated within the scopeof embodiments described herein are surfactants covalently attached topeptides that comprise modified amino acids and/or peptide analogsdescribed above. Certain non-natural amino acids are shown below.

As used herein, “opioid peptides” are short sequences of amino acidsthat bind to opioid receptors in the body. In some embodiments, opioidpeptides are endogenous peptides, such as, for example, endorphins,enkephalins, endomorphins, dermorphins or the like. In some embodiments,opioid peptides are derived from endogenous opioid peptides (e.g.,pseudo-peptides, constrained peptides, alpha-methyl analogs, or thelike). In some embodiments, opioid peptides are exogenous and/orsynthetic and comprise modified amino acids and/or unnatural amino acidsthat mimic the effects of opioid peptides.

As used herein, the term “variant” is interpreted to mean a peptide thatdiffers from a reference peptide, but retains essential properties. Atypical variant of a peptide differs in amino acid sequence fromanother, reference peptide. Generally, differences are limited so thatthe sequences of the reference peptide and the variant are closelysimilar overall and, in many regions, identical. A variant and referencepeptide may differ in amino acid sequence by one or more substitutions,additions, deletions in any combination. A substituted or inserted aminoacid residue may or may not be one encoded by the genetic code.Non-naturally occurring variants of peptides may be made by mutagenesistechniques, by direct synthesis, and by other suitable recombinantmethods.

Reference now will be made in detail to various embodiments andparticular applications of the covalently modified peptides and/orproteins described herein. While the covalently modified peptides and/orproteins will be described in conjunction with the various embodimentsand applications, it will be understood that such embodiments andapplications are exemplary and are not intended to limit the scope ofthe embodiments described herein. In addition, throughout thisdisclosure various patents, patent applications, websites andpublications are referenced, and unless otherwise indicated, each isincorporated by reference in for relevant disclosure referenced herein.

Peptides

There are many important roles played by peptides in the body and somecommercial opportunities have been exploited (Nestor, J. J., Jr. (2009)Current Medicinal Chemistry 16: 4399-4418; Stevenson, C. L. (2009) CurrPharm Biotechnol 10: 122-137). However even these recognized targets(Tyndall, J. D., et al. (2005) Chem Rev 105: 793-826) and productscontinue to suffer from deficiencies in duration of action andbioavailability. In some embodiments, the improved peptides describedherein provide longer duration of action and/or bioavailability and/ortherapeutic efficacy compared to currently available commercialproducts. Some illustrative examples of peptides that representattractive commercial targets for analog design (agonists andantagonists) for clinical development include, for example, members ofthe Class B, G Protein-Coupled Receptor (GPCR) ligands and relatedpeptides (“Secretin family”): Secretin, Parathyroid Hormone (PTH),Parathyroid Hormone-related Protein (PTHrP), Glucagon, Glucagon LikeProtein-1 and -2 (GLP-1, GLP-2), Glucose-dependent InsulinotropicPeptide (GIP), Oxyntomodulin, Pituitary Adenylate Cyclase-ActivatingPeptide (PACAP), Vasoactive Intestinal Peptide (VIP), Amylin (andanalogs such as pramlintide, devalintide, et al.), Calcitonin (andanalogs such as salmon calcitonin, elcatonin, et al.), calcitoningene-related peptide (CGRP), Adrenomedulin, Corticotrophin-ReleasingFactor family (CRF, Xerecept; Urocortin), and the like, includingsynthetic analogs thereof which would be improved as clinical productsthrough further modification by the methods described herein. Alsocontemplated within the scope of embodiments presented herein are Opioidpeptide families; such peptides would benefit from the methods ofpeptide modification described herein to give increased duration ofaction and increased specificity. By way of example analogs of theendomorphin, dynorphin, enkephalin, dermorphin, casomorphin, et al.peptide families offer attractive therapeutic approaches to thetreatment of pain, addiction, et al. Additional attractive peptidetargets for modification to yield peptide products described herein arethe hypothalamic hormones, for example gonadotrophin hormone-releasinghormone and its analogs (for example nafarelin, goserelin, triptorelin,leuprorelin, fertirelin, histrelin, buserelin, ganirelix, cetrorelix,degarelix, deslorelin and the like), adrenocorticotrophin, somatostatin(for example octreotide, lanreotide, valpreotide et al.),thyrotropin-releasing hormone, growth hormone-releasing hormone, andneurotensin. A further example of attractive commercial targets are thepituitary hormones and analogs such as vasopressin (desmopressin, andthe like), oxytocin and analogs, thyroid hormone stimulating hormone,prolactin, growth hormone, luteinizing hormone, follicular-stimulatinghormone, alpha melanocyte-stimulating hormone analogs (melanotananalogs), and the like, as well as their analogs. Growth factors are animportant class of molecules that may be advantageously modified usingthe methods described herein to yield improved pharmaceuticalcandidates, for example insulin (and analogs such as Lispro, Levemir,glargin, et al.), insulin-like growth factor-I (IGF-I or Somatomedin-C),Nerve Growth Factor (NGF), Fibroblast Growth Factor (FGF; FGF-18,FGF-20, FGF-21, and the like), Keratinocyte Growth Factor (KGF) andVascular Endothelial Growth Factor (VEGF), and the like. Especiallyattractive targets are those peptides which control gut function andappetite, but which have short duration of action (some of which arementioned above), including but not limited to, ghrelin, PancreaticPeptide, Peptide YY, Neuropeptide Y, Cholecystokinin (Sincalide, etal.), Melanocortin, and the like. Additional targets benefiting from themethods described herein are the proinflammatory adipose tissue productsrelating to obesity, including Leptin (and related analogs such as OB-3peptide), adipokines, adiponectin, Chemerin, visfatin, nesfatin,resistin, tumor necrosis factor alpha, chemokines, monocyte chemotacticprotein-1 (MCP-1), omentin, interleukins, and the like. Importanttargets that would benefit from improved pharmaceutical andpharmacodynamic behavior are proteins that control immune function,among which are given the following examples, which are not meant to belimiting, merely illustratory: members of the interferon family(interferons-alpha, -beta, -gamma, -kappa, -omega, the IL-10 cytokinefamily, including IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, and thelike), Thymopentin, Thymosin alpha1, and the like. Important peptideproducts which control the circulatory, or blood clotting would beimproved by the methods described herein whereby increased duration ofaction would be achieved, for example, bivalirudin (Angiomax),eptifibatide (Integrelin), atrial natriuretic peptides (ANP, Ularitide),brain natriuretic peptide, c type natriuretic peptide, b-typenatriuretic peptide (nesiritide), angiotensin, Angiostatin, Rotigaptide,thrombospondins and the like. Peptides that stimulate stem cellproliferation and differentiation are important agents that could beimproved by the modifications described herein. For example,erythropoietin, hematide, thrombopoietin, macrophage colony-stimulatingfactor (M-CSF), leukemia inhibitory factor (LIF), interleukin-6 (IL-6)),granulocyte colony-stimulating factor (G-CSF),granulocyte-macrophage-colony-stimulating factor (GM-CSF), and the likeare contemplated as peptides that are suitable for covalent attachmentto a surfactant (e.g., an alkyl glycoside surfactant). Neurotrophicfactors are another class of small proteins that would greatly benefitfrom modifications described herein to increase duration of action andefficacy. For example, Glial cell-derived neurotrophic factor (GDNF) andfamily (neurturin, artemin, persephin), neurotrophins such as nervegrowth factor (NGF), BDNF and the like. Proinflammatory and pain-causingpeptides are important peptide targets that would be improved bymodifications described herein. For example, inhibitors of bradykinin(or its release, e.g. ecallantide) and substance P especiallyantagonists, offer important therapeutic targets (icatibant, et al.) aresuitable for covalent attachment of surfactants (e.g. alkyl-glycosidesurfactants). Inhibitors of viral fusion (Fuzeon), protein maturation(protease inhibitors) or integration suffer from short duration ofaction. Modification of such inhibitors via covalent attachment of asurfactant (e.g., an alkyl-glycoside surfactant) will allow for longerduration of action. Many proteases have been found to be involved indisease and inhibitors of their action have reached the clinic(Abbenante, G. and Fairlie, D. P. (2005) Med Chem 1: 71-104), but wouldbe further improved by the modifications described herein and suchtargets are also contemplated within the scope of the embodimentsdescribed herein. Additional natural peptide products and analogsthereof such as conotoxin peptides for pain, antimicrobial defensins andthe like also suffer from lack of bioavailability and short duration ofaction in physiological fluid and therefore would benefit from thepeptide modifications described herein. Those skilled in the art willrecognize many additional commercially important peptides that areamenable to modifications described herein and provide increasedduration of action and bioavailability, and such peptides are alsocontemplated within the scope of the present disclosure.

Modifications at the amino or carboxyl terminus may optionally beintroduced into the present peptides (Nestor, J. J., Jr. (2009) CurrentMedicinal Chemistry 16: 4399-4418). For example, the present peptidescan be truncated or acylated on the N-terminus to yield peptidesexhibiting low efficacy, partial agonist and antagonist activity, as hasbeen seen for some peptides (Gourlet, P., et al. (1998) Eur J Pharmacol354: 105-111, Gozes, I. and Furman, S. (2003) Curr Pharm Des 9:483-494), the contents of which is incorporated herein by reference).Other modifications to the N-terminus of peptides, such as deletions orincorporation of D-amino acids such as D-Phe also can give potent andlong acting agonists or antagonists when substituted with themodifications described herein such as long chain alkyl glycosides. Suchagonists and antagonists also have commercial utility and are within thescope of contemplated embodiments described herein.

Aside from the twenty standard amino acids, there are a vast number of“nonstandard amino acids” or unnatural amino acids that are known to theart and that may be incorporated in the compounds described herein, asdescribed above. Other nonstandard amino acids are modified withreactive side chains for conjugation (Gauthier, M. A. and Klok, H. A.(2008) Chem Commun (Camb) 2591-2611; de Graaf, A. J., et al. (2009)Bioconjug Chem 20: 1281-1295). In one approach, an evolved tRNA/tRNAsynthetase pair and is coded in the expression plasmid by the ambersuppressor codon (Deiters, A, et al. (2004). Bio-org. Med. Chem. Lett.14, 5743-5). For example, p-azidophenylalanine was incorporated intopeptides and then reacted with a functionalized surfactant, or a PEGpolymer having an acetylene moiety in the presence of a reducing agentand copper ions to facilitate an organic reaction known as “Huisgen[3+2] cycloaddition.” A similar reaction sequence using the reagentsdescribed herein containing an acetylene modified alkyl glycoside or PEGmodified glycoside will result in PEGylated or alkyl glycoside modifiedpeptides. For peptides of less than about 50 residues, standard solidphase synthesis is used for incorporation of said reactive amino acidresidues at the desired position in the chain. Such surfactant-modifiedpeptides and/or proteins offer a different spectrum of pharmacologicaland medicinal properties than peptides modified by PEG incorporationalone.

Intermediates

In one embodiment provided herein are intermediates and/or reagentscomprising a surfactant moiety and a reactive functional group capableof forming a bond with a reactive functional group on a natural orunnatural amino acid. These intermediates and/or reagents allow forimprovement in the bioavailability and pharmaceutical, pharmacokineticand/or pharmacodynamic behavior of peptides and/or proteins of use inhuman and animal disease. Covalent attachment of such intermediatesand/or reagents via a functional group on a side chain of an amino acid,for example on an epsilon-amino function of Lys, the sulfhydryl of Cys,or at the amino or carboxy terminus of the peptide and/or protein targetallows for synthesis of the peptide products described herein. Inspecific embodiments, non-ionic surfactant moieties are mono ordisaccharides with an O-alkyl glycosidic substitution, said glycosidiclinkage being of the alpha or beta configuration. In specificembodiments, O-alkyl chains are from C₁-C₂₀ or from C₆-C₁₆ alkyl chains.

In another embodiment provided herein are intermediates and/or reagentscomprising a non-ionic surfactant moiety with certain alkyl glycosidiclinkage that mimic O-alkyl glycosidic linkages and a reactive functionalgroup capable of forming a bond with a reactive functional group on anatural or unnatural amino acid. Such intermediates and/or reagentscontain S-linked alkyl chains or N-linked alkyl chains and have alteredchemical and/or enzymatic stability compared to the O-linked alkylglycoside-linked products.

In some embodiments, an intermediate and/or reagent provided herein is acompound wherein the hydrophilic group is a modified glucose, galactose,maltose, glucuronic acid, diglucuronic acid, maltouronic acid or thelike. In some embodiments, the hydrophilic group is glucose, maltose,glucuronic acid, diglucuronic acid or maltouronic acid and thehydrophobic group is a C₁-C₂₀ alkyl chain or an aralkyl chain. In someembodiments the glycosidic linkage to the hydrophobic group is of analpha configuration and in some the linkage is beta at the anomericcenter on the saccharide.

In some embodiments, the hydrophilic group is glucose, maltose,glucuronic acid, diglucuronic acid or maltouronic acid and thehydrophobic group is a C₁-C₂₀ alkyl or aralkyl chain.

In some embodiments, an intermediate and/or reagent provided hereincomprises a surfactant containing a reactive functional group that is acarboxylic acid group, an amino group, an azide, an aldehyde, amaleimide, a sulfhydryl, a hydroxylamino group, an alkyne or the like.

In some embodiments, the intermediate and/or reagent is an O-linkedalkyl glycoside with one of the hydroxyl functions modified to be acarboxylic acid or amino functional group. In some embodiments, thereagent is a 1-O-alkyl glucuronic acid of alpha or beta configurationand the alkyl chain is from C₁ to C₂₀ in length. In some of suchembodiments, the alkyl group is from C₆ to C₁₈ in length. In some ofsuch embodiments, the alkyl group is from C₆ to C₁₆ in length.

In some embodiments, the reagent comprises a 1-O-alkyl diglucuronic acidof alpha or beta configuration and the alkyl chain is from C₁ to C₂₀ inlength. In some of such embodiments, the alkyl group is from C₆ to C₁₆in length.

In some embodiments, the reagent is an S-linked alkyl glycoside of alphaor beta configuration with one of the hydroxyl functions modified to bea carboxylic acid or amino functional group.

In some embodiments, the reagent is an N-linked alkyl glycoside of alphaor beta configuration with one of the hydroxyl functions modified to bea carboxylic acid or amino functional group.

In yet another embodiment provided herein are peptide and/or proteinproducts containing a covalently linked alkyl glycoside with propertiesacceptable for use in human and animal disease. Scheme 1 lists exemplarynon-ionic surfactants that can be modified to yield the reagents and/orintermediates that are useful for synthesis of surfactant-modifiedpeptide products described herein.

In some embodiments, the covalently modified peptides and/or proteinsdescribed herein incorporate a surfactant moiety into the peptidestructure. In specific embodiments, the covalently modified peptidesand/or proteins described herein incorporate a non-ionic surfactant ofthe alkyl, alkoxyaryl, or aralkyl glycoside class. Alkyl glycosides, forexample in the mono and disaccharide classes, are important commoditiesand are widely used in the food, service and cleaning industries. Thustheir production on commercially significant scale has been the subjectof extensive study. Both enzymatic and chemical processes are availablefor their production at very low cost (Park, D. W., et al. (2000)Biotechnology Letters 22: 951-956). These alkyl glycosides can bemodified further to generate the intermediates for the synthesis of thecovalently modified peptides and/or proteins described herein. Thus itis known that 1-dodecyl beta-D-glucoside is preferentially oxidized onthe 6-position to yield the corresponding glucuronic acid analog in highyield when using the unprotected material and platinum black catalyst inthe presence of oxygen (van Bekkum, H. (1990) Carbohydrates as OrganicRaw Materials 289-310). Additional chemoselective methods for oxidationof the primary alcohol at the 6 position of alkyl glucosides areavailable. For example, use of catalytic amounts of2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO) with stoichiometricamounts of the organic oxidant [bis(acetoxy)iodo]benzene (BAIB) (DeMico, A., et al. (1997) J Org Chem 1997: 6974-6977) gave outstandingyields of nucleoside-5′-carboxylic acids (Epp, J. B. and Widlanski, T.S. (1999) J Org Chem 64: 293-295) by oxidation of the primary hydroxyl.This oxidation is chemoselective for the primary hydroxyl even when theother, secondary hydroxyls are unprotected (Codee, J. D., et al. (2005)J Am Chem Soc 127: 3767-3773). In a similar manner, 1-dodecylβ-D-glucopyranoside and 1-tetradecyl β-D-glucopyranoside were oxidizedto the corresponding uronic acids (1-dodecyl β-D-glucuronic acid,1-tetradecyl β-D-glucuronic acid) by oxidation with TEMPO using KBr andsodium hypochlorite as stoichiometric oxidant (Milkereit, G., et al.(2004) Chem Phys Lipids 127: 47-63) in water. A mild oxidation procedureusing (diacetoxyiodo)benzene (DAIB aka BAIB) is given in the Examples.Certain of these glucuronic acid intermediates are commerciallyavailable (for example octyl b-D-glucuronic acid; Carbosynth, Mo. 07928)and, as indicated, a broad range are subject to preparation by routinemethods (Schamann, M. and Schafer, H. J. (2003) Eur J Org Chem 351-358;Van den Bos, L. J., et al. (2007) Eur J Org Chem 3963-3976) or uponrequest. Scheme 2 illustrates, as examples, certain functionalizedsurfactant intermediates comprising a —COOH group as a reactivefunctional group that are used to prepare the intermediates and/orreagents described herein.

Similarly, aralkyl glycosides (including alkoxyaryl) can form the basisfor closely related nonionic surfactant reagents. For example,4-alkoxyphenyl β-D-glucopyranosides are readily synthesized by thereaction of 4-alkyloxyphenols with penta-O-acetyl β-D-glucose in thepresence of boron trifluoride etherate. Subsequent deacetylation usingtrimethylamine in methanol/water and selective oxidation as describedabove and in the examples, yields the alkoxyaryl glucuronic acidreagents suitable for forming the reagents and peptides described herein((Smits, E., et al. (1996) J Chem Soc, Perkin Trans 1 2873-2877; Smits,E., et al. (1997) Liquid Crystals 23: 481-488).

The glucuronic acid class of intermediate is readily activated bystandard coupling agents for linkage to an amino acid side chain, e.g.that of Lys. Thus Fmoc-Lys-O-Tms (trimethylsilyl=TMS) can be reactedwith octyl beta-D-glucuronic acid in the presence of a coupling agentand the O-Tms protecting group can then be hydrolyzed on aqueous workupto yield Fmoc-Lys(1-octyl beta-D-glucuronamide) as shown in Scheme 4.This reagent can be used for incorporation into the solid phasesynthesis of peptides, using standard coupling protocols, when it isdesired to incorporate the surfactant moiety near the N-terminal regionof the molecule. The secondary hydroxyl groups can be left unprotected,due to the very much higher reactivity of the Lys amino functional groupor they can be protected by peracetylation. If an acetyl protected formis used, the acetyl protecting groups can be removed in high yield bytreatment with either MeOH/NaOMe or by MeOH/Et₃N. Scheme 4 illustratespreparation of the reagents described herein.

In some embodiments, reagents and/or intermediates for the preparationof the biologically active peptide products described herein comprise afamily of surfactant-modified linker amino acids for incorporation intosynthetic peptide products. Thus in one embodiment, peptide productsdescribed herein are synthesized in a linear fashion wherein afunctionalized surfactant is attached to a reversibly-protected linkeramino acid via a functional group on a side chain of a linker amino acid(e.g., an amino group of a lysine residue) to yield a proprietaryreagent (as shown in Scheme 4.) which can be incorporated into thegrowing peptide chain and then the remaining peptide is synthesized byattachment of further amino acids to the cysteine residue. Protectinggroup suitable for synthesis of modified peptides and/or proteindescribed herein are described in, for example, T. W. Green, P. G. M.Wuts, Protective Groups in Organic Synthesis, Wiley-Interscience, NewYork, 1999, 503-507, 736-739, which disclosure is incorporated herein byreference.

In another embodiment, peptide products described herein are synthesizedby covalent attachment of a functionalized surfactant to a full-lengthpeptide via a suitable functional group on a linker amino acid that isin the peptide chain.

Alternatively a functionalized surfactant can be added to a linker aminoacid side chain which has been deprotected during the course of thesolid phase synthesis of the peptide. As an example, an alkyl glucuronylgroup can be added directly to a linker amino acid side chain (e.g., adeprotected Lys side chain) during the solid phase synthesis of thepeptide. For example, use of Fmoc-Lys(Alloc)-OH as a subunit providesorthogonal protection that can be removed while the peptide is still onthe resin. Thus deprotection of the Lys side chain usingPd/thiobarbital, Pd/1,3-dimethyl barbituric acid (DMBA) or other allocdeprotection recipe allows exposure of the amino group for coupling withthe acyl protected or unprotected 1-octyl beta-D-glucuronic acid unit orfor side chain lactam formation. Final deprotection with a low % CF₃CO₂H(TFA) cleavage cocktail will then deliver the desired product. Althoughthe glycosidic linkage is labile to strong acid, the experience here andby others is that it is relatively stable to low % TFA cleavageconditions. Alternatively, acyl protection (e.g. acetyl, Ac; benzoyl,Bz) or trialkylsilyl protection on the saccharide OH functional groupsmay be used to provide increased protection to the glycosidic linkage.Subsequent deprotection by base (NH₂NH₂/MeOH; NH₃/MeOH, NaOMe/MeOH)yields the desired deprotected product. Scheme 4 illustrates reagentsdescribed herein. Scheme 5 illustrates peptide intermediates describedherein.

Additional reagents are generated by modification of the 6-positionfunctional group to give varied means of linkage to amino acid sidechain functional groups, as shown below in Scheme 6. Thus aminosubstitution can be used for linkage to Asp or Glu side chains. Azido oralkyne substitution can be used for linkage to unnatural amino acidscontaining the complementary acceptor for Huisgen 3+2 cycloaddition(Gauthier, M. A. and Klok, H. A. (2008) Chem Commun (Camb) 2591-2611).Aminoxy or aldehyde functional groups can be used to link to aldehyde(i.e. oxime linkage) or to amino functions (i.e. reductive alkylation),respectively. The maleimide or —NH—(C═O)—CH₂—Br functional group canbind chemoselectively with a Cys or other SH functional group. Thesetypes of linkage strategies are advantageous when used in conjunctionwith the reagents described herein. Interconversion of functional groupsis widely practiced in organic synthesis and comprehensive lists ofmultiple routes to each of the functional group modifications listedherein are available (Larock, R. C. (1999)) “Comprehensive OrganicTransformations”, VCH Publishers, New York.

Thus, for example, the primary hydroxyl on position 6 of octyl1-β-D-glucoside is converted to the azide by activation and displacementwith an azide anion, reactions such as reactions used in carbohydratechemistry (e.g. by tosylation followed by NaN₃). The corresponding azideis reduced to the amino function by reduction with thiolacetic acid inpyridine (Elofsson, M., et al. (1997) Tetrahedron 53: 369-390) or bysimilar methods of amino group generation (Stangier, P., et al. (1994)Liquid Crystals 17: 589-595). Approaches to the acetylene, aminoxy, andaldehyde moieties are best carried out on the triacetoxy form, availablefrom the commercially available glucoside by treatment with Ac₂O,followed by mild hydrolysis of the primary amine. This 6-hydroxy formcan be selectively oxidized to the aldehyde, or activated as a tosylateor triflate and displaced by NH₂OH or by sodium acetylide. The maleimidelinkage can be through a carbon linkage as shown or, preferably thoughan O or amide linkage, again by displacement of the activated hydroxylor coupling of the glucuronic acid derivative to an amino linkedmaleimide reagent, well known in the art. Additional functional groupinterconversions are well known to those of average skill in the art ofmedicinal chemistry and are within the scope of the embodimentsdescribed herein.

Also contemplated within the scope of synthetic methods described hereinare surfactants wherein the saccharide and hydrophobic chain arecovalently attached via an alpha glycosidic linkage. Synthetic routes topredominantly α-linked glycosides are well known in the art andtypically originate with the peracetyl sugar and use acidic catalysis(e.g. SnCl₄, BF₃ or HCl) to effect the α-glycosylation (Cudic, M. andBurstein, G. D. (2008) Methods Mol Biol 494: 187-208, Vill, V., et al.(2000) Chem Phys Lipids 104: 75-91, incorporated herein by reference forsuch disclosure). Similar synthetic routes exist for disaccharideglycosides (von Minden, H. M., et al. (2000) Chem Phys Lipids 106:157-179, incorporated herein by reference for such disclosure).Functional group interconversions then proceed as above to lead to the6-carboxylic acid, et al. for generation of the corresponding α-linkedreagents.

Scheme 6 lists certain compounds and reagents useful in the synthesis ofthe covalently modified peptides and/or proteins described herein.Standard nomenclature using single letter abbreviations for amino acidsare used.

Many alkyl glycosides can be synthesized by known procedures, asdescribed, e.g., in (Rosevear, P., et al. (1980) Biochemistry 19:4108-4115, Li, Y. T., et al. (1991) J Biol Chem 266: 10723-10726) orKoeltzow and Urfer, J. Am. Oil Chem. Soc., 61:1651-1655 (1984), U.S.Pat. No. 3,219,656 and U.S. Pat. No. 3,839,318 or enzymatically, asdescribed, e.g., in (Li, Y. T., et al. (1991) J Biol Chem 266:10723-10726, Gopalan, V., et al. (1992) J Biol Chem 267: 9629-9638).O-alkyl linkages to natural amino acids such as Ser can be carried outon the Fmoc-Ser-OH using peracetylglucose to yieldNα-Fmoc-4-O-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-L-serine. Thismaterial is selectively deprotected at the primary carbon atom (position6) and selectively oxidized using TEMPO/BAIB as described above to yieldthe corresponding 6-carboxyl function which may be coupled to lipophilicamines to generate a new class of nonionic surfactant and reagents(Scheme 7).

The linkage between the hydrophobic alkyl and the hydrophilic saccharidecan include, among other possibilities, a glycosidic, thioglycosidic,amide (Carbohydrates as Organic Raw Materials, F. W. Lichtenthaler ed.,VCH Publishers, New York, 1991), ureide (Austrian Pat. 386,414 (1988);Chem. Abstr. 110:137536p (1989); see Gruber, H. and Greber, G.,“Reactive Sucrose Derivatives” in Carbohydrates as Organic RawMaterials, pp. 95-116) or ester linkage (Sugar Esters: Preparation andApplication, J. C. Colbert ed., (Noyes Data Corp., New Jersey), (1974)).

Examples from which useful alkyl glycosides can be chosen formodification to the reagents or for the formulation of the productsdescribed herein, include: alkyl glycosides, such as octyl-, nonyl-,decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl, pentadecyl-,hexadecyl-, heptadecyl-, and octadecyl-D-maltoside, -glucoside or-sucroside (i.e., sucrose ester) (synthesized according to Koeltzow andUrfer; Anatrace Inc., Maumee, Ohio; Calbiochem, San Diego, Calif.; FlukaChemie, Switzerland); alkyl thiomaltosides, such as heptyl, octyl,dodecyl-, tridecyl-, and tetradecyl-β-D-thiomaltoside (synthesizedaccording to Defaye, J. and Pederson, C., “Hydrogen Fluoride, Solventand Reagent for Carbohydrate Conversion Technology” in Carbohydrates asOrganic Raw Materials, 247-265 (F. W. Lichtenthaler, ed.) VCHPublishers, New York (1991); Ferenci, T., J. Bacteriol, 144:7-11(1980)); alkyl thioglucosides, such as 1-dodecyl- or 1-octyl-thio α- orβ-D-glucopyranoside (Anatrace, Inc., Maumee, Ohio; see Saito, S. andTsuchiya, T. Chem. Pharm. Bull. 33:503-508 (1985)); alkyl thiosucroses(synthesized according to, for example, Binder, T. P. and Robyt, J. F.,Carbohydr. Res. 140:9-20 (1985)); alkyl maltotriosides (synthesizedaccording to Koeltzow and Urfer); long chain aliphatic carbonic acidamides of sucrose amino-alkyl ethers; (synthesized according to AustrianPatent 382,381 (1987); Chem. Abstr., 108:114719 (1988) and Gruber andGreber pp. 95-116); derivatives of palatinose and isomaltamine linked byamide linkage to an alkyl chain (synthesized according to Kunz, M.,“Sucrose-based Hydrophilic Building Blocks as Intermediates for theSynthesis of Surfactants and Polymers” in Carbohydrates as Organic RawMaterials, 127-153); derivatives of isomaltamine linked by urea to analkyl chain (synthesized according to Kunz); long chain aliphaticcarbonic acid ureides of sucrose amino-alkyl ethers (synthesizedaccording to Gruber and Greber, pp. 95-116); and long chain aliphaticcarbonic acid amides of sucrose amino-alkyl ethers (synthesizedaccording to Austrian Patent 382,381 (1987), Chem. Abstr., 108:114719(1988) and Gruber and Greber, pp. 95-116).

Some preferred glycosides include the saccharides maltose, sucrose,glucose and galactose linked by glycosidic or ester linkage to an alkylchain of 6, 8, 10, 12, or 14 carbon atoms, e.g., hexyl-, octyl-, decyl-,dodecyl- and tetradecyl-maltoside, sucroside, glucoside and galactoside.In the body these glycosides are degraded to non-toxic alcohol or fattyacid and an oligosaccharide or saccharide. The above examples areillustrative of the types of alkyl glycosides to be used in the methodsclaimed herein, however the list is not intended to be exhaustive.

Generally, these surfactants (e.g., alkyl glycosides) are optionallydesigned or selected to modify the biological properties of the peptide,such as to modulate bioavailability, half-life, receptor selectivity,toxicity, biodistribution, solubility, stability, e.g. thermal,hydrolytic, oxidative, resistance to enzymatic degradation, and thelike, facility for purification and processing, structural properties,spectroscopic properties, chemical and/or photochemical properties,catalytic activity, redox potential, ability to react with othermolecules, e.g., covalently or noncovalently, and the like.

Surfactants

The term “surfactant” comes from shortening the phrase “surface activeagent”. In pharmaceutical applications, surfactants are useful in liquidpharmaceutical formulations in which they serve a number of purposes,acting as emulsifiers, solubilizers, and wetting agents. Emulsifiersstabilize the aqueous solutions of lipophilic or partially lipophilicsubstances. Solubilizers increase the solubility of components ofpharmaceutical compositions increasing the concentration which can beachieved. A wetting agent is a chemical additive which reduces thesurface tension of a fluid, inducing it to spread readily on a surfaceto which it is applied, thus causing even “wetting” of the surface withthe fluids. Wetting agents provide a means for the liquid formulation toachieve intimate contact with the mucous membrane or other surface areaswith which the pharmaceutical formulation comes in contact. Thussurfactants may be useful additives for stabilization of the formulationof the peptide products described herein as well as for the modificationof the properties of the peptide itself.

In specific embodiments, alkyl glycosides which are syntheticallyaccessible, e.g., the alkyl glycosides dodecyl, tridecyl and tetradecylmaltoside or glucoside as well as sucrose dodecanoate, tridecanoate, andtetradecanoate are suitable for covalent attachment to peptides asdescribed herein. Similarly, the corresponding alkylthioglycosides arestable, synthetically accessible surfactants which are acceptable forformulation development.

A wide range of physical and surfactant properties can be achieved byappropriate modification of the hydrophobic or hydrophilic regions ofthe surfactant (e.g., the alkyl glycoside). For example, a studycomparing the bilayer activity of dodecyl maltoside (DM) with that ofdodecyl glucoside (DG) found that of DM to be more than three timeshigher than that of DG, despite having the same length of hydrophobictail (Lopez, O., et al. (2002) Colloid Polym Sci 280: 352-357). In thisparticular instance the identity of the polar region (disaccharide vsmonosaccharide) influences surfactant behavior. In the case of asurfactant linked to a peptide, e.g. the peptide products describedherein, the peptide region also may contribute hydrophobic orhydrophilic character to the overall molecule. Thus tuning of thephysical and surfactant properties may be used to achieve the particularphysical and pharmaceutical properties suitable for the individualpeptide targets.

PEG Modification

In some embodiments, surfactant-modified peptide products describedherein are further modified to incorporate one or more PEG moieties(Veronese, F. M. and Mero, A. (2008) BioDrugs 22: 315-329). In someinstances, incorporation of large PEG chains prevents filtration of thepeptide in the glomeruli in the kidney into the dilute urine formingthere (Nestor, J. J., Jr. (2009) Current Medicinal Chemistry 16:4399-4418, Caliceti, P. and Veronese, F. M. (2003) Adv Drug Deliv Rev55: 1261-1277). In some embodiments, an optional PEG hydrophilic chainallows for balancing the solubility and physical properties of thepeptides or proteins that have been rendered hydrophobic by theincorporation of the longer chain alkyl glycoside moiety.

PEGylation of a protein can have potentially negative effects as well.It is widely recognized that PEGylation and other polymericmodifications can cause a substantial loss of biological activity formany proteins and this may relate to ligands for specific classes ofreceptors. In such instances there may be a benefit to reversiblePEGylation (Peleg-Shulman, T., et al. (2004) J Med Chem 47: 4897-4904,Greenwald, R. B., et al. (2003) Adv Drug Deliv Rev 55: 217-250, Roberts,M. J. and Harris, J. M. (1998) J Pharm Sci 87: 1440-1445).

In addition, the increased molecular mass may prevent penetration ofphysiological barriers other than the glomerular membrane barrier. Forexample, it has been suggested that high molecular weight forms ofPEGylation may prevent penetration to some tissues and thereby reducetherapeutic efficacy. In addition, high molecular weight may preventuptake across mucosal membrane barriers (nasal, buccal, vaginal, oral,rectal, lung delivery). However delayed uptake may be highlyadvantageous for administration of stable molecules to the lung,substantially prolonging the duration of action. The peptide and/orprotein products described herein have increased transmucosalbioavailability and this will allow longer chain PEG modifications to beused in conjunction with the surfactant modification with theachievement of commercially significant bioavailability followingintranasal or other transmucosal route.

In some embodiments, long chain PEG polymers, and short chain PEGpolymers are suitable for modification of the proteins and peptidesdescribed herein. Administration of treatments for diabetes byinhalation is a new approach for drug delivery and the lung has a highlypermeable barrier (e.g. Exubera). For this application, delayedpenetration of the lung barrier, preferred forms of PEGylation are inthe lower molecular weight range of C₁₀ to C₄₀₀ (roughly 250 to 10,000Da). Thus while a primary route to prolongation by PEG is theachievement of an “effective molecular weight” above the glomerularfiltration cut-off (greater than 68 kDa), use of shorter chains may be aroute for prolongation of residence in the lung for treatment of lungdiseases and other respiratory conditions. Thus PEG chains of about 500to 3000 Da are of sufficient size to slow the entry into the peripheralcirculation, but insufficient to cause them to have a very prolongedcirculation time. In some embodiments, PEGylation is applied to giveincreased local efficacy to the lung tissue with reduced potential forsystemic side effects for the the covalently modified peptides and/orproteins described herein. In some of such embodiments, PEG chains inthe range from about 750 to about 1500 Da are referred collectively as“PEG1K.”

In addition, other polymers may be used in conjunction with thecompounds of described herein in order to optimize their physicalproperties. For example poly(2-ethyl 2-oxazoline) conjugates havevariable hydrophobicity and sufficient size to enhance duration ofaction (Mero, A., et al. (2008) J Control Release 125: 87-95). Linkageof such a polymer to a saccharide yields a class of surfactant suitablefor use in modification of peptides and/or proteins described herein.

Polyethylene glycol chains are functionalized to allow their conjugationto reactive groups on the peptide and/or protein chain. Typicalfunctional groups allow reaction with amino, carboxyl or sulfhydrylgroups on the peptide through the corresponding carboxyl, amino ormaleimido groups (and the like) on the polyethylene glycol chain. In anembodiment, PEG comprises a C₁₀-C₃₀₀₀ chain. In another embodiment, PEGhas a molecular weight above 40,000 Daltons. In yet another embodiment,PEG has a molecular weight below 10,000 Daltons. PEG as a proteinmodification is well known in the art and its use is described, forexample, in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;4,791,192; and 4,179,337.

A non-traditional type of PEG chain is modified to be amphiphilic innature. That is it has both the hydrophilic PEG structure but ismodified to contain hydrophobic regions such as fatty acid esters andother hydrophobic components. See for example (Miller, M. A., et al.(2006) Bioconjug Chem 17: 267-274); Ekwuribe, et al. U.S. Pat. No.6,309,633; Ekwuribe, et al. U.S. Pat. No. 6,815,530; Ekwuribe, et al.U.S. Pat. No. 6,835,802). Although these amphiphilic PEG conjugates toproteins were originally developed to increase oral bioavailability theywere relatively ineffective in this role. However the use of suchamphiphilic PEG conjugates with amphipathic peptides will givesignificantly prolonged residence in the lung to extend the usefulbiological activity of these pharmaceuticals. The preferred PEG chainsare in the molecular weight range of 500 to 3000 Da. Detaileddescriptions of the methods of synthesis of these conjugates is given inthe references above, the full content of which is incorporated herein.

A PEG entity itself does not have a functional group to be attached to atarget molecular, such as a peptide. Therefore, to create PEGattachment, a PEG entity must be functionalized first, then afunctionalized attachment is used to attach the PEG entity to a targetmolecule, such as a peptide (Greenwald, R. B., et al. (2003) Adv DrugDeliv Rev 55: 217-250, Veronese, F. M. and Pasut, G. (2005) Drug DiscovToday 10: 1451-1458, Roberts, M. J., et al. (2002) Adv Drug Deliv Rev54: 459-476). In one embodiment, site-specific PEGylation can beachieved through Cys substitution on a peptide molecule. The targetpeptide can be synthesized by solid phase synthesis, recombinant means,or other means, as described herein.

Thus in some embodiments, a peptide product described herein comprises aLys or other reactive residue modified with an alkyl glycoside andspecific PEGylation on at least one Cys residue, a Lys residue or otherreactive amino acid residue elsewhere in the molecule.

In another embodiment, a Lys or other residue residue with anucleophilic side chain may be used for incorporation of the PEGresidue. This may be accomplished through the use of an amide orcarbamate linkage to a PEG-carboxyl or PEG-carbonate chain. See forexample as described (Veronese, F. M. and Pasut, G. (2005) Drug DiscovToday 10: 1451-1458). An alternative approach is to modify the Lys sidechain amino function through attachment of an SH containing residue,such as mercaptoacetyl, mercaptopropionyl (CO—CH₂—CH₂—CH₂—SH), and thelike. Alternatively the PEG chain may be incorporated at the C-Terminusas an amide during the course of the synthesis. Additional methods forattaching PEG chains utilize reaction with the side chains of His andTrp. Other similar methods of modifying the peptide chain to allowattachment of a PEG chain are known in the art and are incorporatedherein by reference (Roberts, M. J., et al. (2002) Adv Drug Deliv Rev54: 459-476).

Formulations

In one embodiment, the covalently modified peptides or proteins asdisclosed herein are provided in a formulation that further reduces,prevents, or lessens peptide and/or protein association or aggregationin the composition, for example, reduces peptide and/or proteinself-association or self-aggregation, or reduces association oraggregation with other peptides or proteins when administered to thesubject.

Self-Association at high protein concentration is problematic intherapeutic formulations. For example, self-association increases theviscosity of a concentrated monoclonal antibody in aqueous solution.Concentrated insulin preparations are inactivated by self aggregation.These self associating protein interactions, particularly at highprotein concentration, reduce, modulate or obliterate biologicalactivity of many therapeutics (Clodfelter, D. K., et al. (1998) PharmRes 15: 254-262). Therapeutic proteins formulated at high concentrationsfor delivery by injection or other means can be physically unstable orbecome insoluble as a result of these protein interactions.

A significant challenge in the preparation of peptide and proteinformulations is to develop manufacturable and stable dosage forms.Physical stability properties, critical for processing and handling, areoften poorly characterized and difficult to predict. A variety ofphysical instability phenomena are encountered such as association,aggregation, crystallization and precipitation, as determined by proteininteraction and solubility properties. This results in significantmanufacturing, stability, analytical, and delivery challenges.Development of formulations for peptide and protein drugs requiring highdosing (on the order of mg/kg) are required in many clinical situations.For example, using the SC route, approximately <1.5 mL is the allowableadministration volume. This may require >100 mg/mL proteinconcentrations to achieve adequate dosing. Similar considerations existin developing a high-concentration lyophilized formulation formonoclonal antibodies. In general, higher protein concentrations permitsmaller injection volume to be used which is very important for patientcomfort, convenience, and compliance. The surfactant-modified compoundsdescribed herein are designed to minimize such aggregation events andmay be further facilitated through the use of small amounts ofsurfactants as herein described.

Because injection is an uncomfortable mode of administration for manypeople, other means of administering peptide therapeutics have beensought. Certain peptide and protein therapeutics may be administered,for example, by intranasal, buccal, oral, vaginal, inhalation, or othertransmucosal administration. Examples are nafarelin (Synarel®) andcalcitonin which are administered as commercial nasal sprayformulations. The covalently modified peptides and/or proteins describedherein are designed to facilitate such transmucosal administration andsuch formulations may be further facilitated through the use of smallamounts of surfactants as described herein.

Typical formulation parameters include selection of optimum solution pH,buffer, and stabilizing excipients. Additionally, lyophilized cakereconstitution is important for lyophilized or powdered formulations. Afurther and significant problem comprises changes in viscosity of theprotein formulation upon self-association. Changes in viscosity cansignificantly alter delivery properties e.g., in spray (aerosol)delivery for intranasal, pulmonary, or oral cavity sprays. Furthermore,increased viscosity can make injection delivery by syringe or iv linemore difficult or impossible.

Many attempts to stabilize and maintain the integrity and physiologicalactivity of peptides have been reported. Some attempts have producedstabilization against thermal denaturation and aggregation, particularlyfor insulin pump systems. Polymeric surfactants are described (Thurow,H. and Geisen, K. (1984) Diabetologia 27: 212-218; Chawla, A. S., et al.(1985) Diabetes 34: 420-424). The stabilization of insulin by thesecompounds was believed to be of a steric nature. Among other systemsused are saccharides (Arakawa, T. and Timasheff, S. N. (1982)Biochemistry 21: 6536-6544), osmolytes, such as amino acids (Arakawa, T.and Timasheff, S. N. (1985) Biophys J 47: 411-414), and water structurebreakers, such as urea (Sato, S., et al. (1983) J Pharm Sci 72:228-232). These compounds exert their action by modulating theintramolecular hydrophobic interaction of the protein or peptide.

Various peptides, peptides, or proteins are described herein and may bemodified with any of the covalently bound surfactant reagents describedherein. Advantageously, the peptide modifications described hereincomprise covalent attachment of a surfactant that comprises bothhydrophilic (e.g. saccharide) and hydrophobic (e.g. alkyl chain) groups,thereby allowing for stabilization of the peptide in physiologicalconditions. In some embodiments, covalent linkage of a moiety comprisinga hydrophilic group and hydrophobic group (e.g. a glycoside surfactant)to a peptide, and/or protein described herein eliminates the need formodifying the amino acid sequence of the peptide, and/or protein toenhance stability (e.g., reduce aggregation).

In some embodiments, the formulations comprise at least one drugcomprising a peptide modified with a surfactant derived reagentdescribed herein and in formulation additionally may be associated witha surfactant, wherein the surfactant is further comprised of, forexample, a saccharide, an alkyl glycoside, or other excipient and can beadministered in a format selected from the group consisting of a drop, aspray, an aerosol, a lyophilizate, a spray dried product, an injectable,and a sustained release format. The spray and the aerosol can beachieved through use of the appropriate dispenser and may beadministered by intranasal, transbuccal, inhalation or othertransmucosal route. The lyophilizate may contain other compounds such asmannitol, saccharides, submicron anhydrous α-lactose, gelatin,biocompatible gels or polymers. The sustained release format can be anocular insert, erodible microparticulates, hydrolysable polymers,swelling mucoadhesive particulates, pH sensitive microparticulates,nanoparticles/latex systems, ion-exchange resins and other polymericgels and implants (Ocusert, Alza Corp., California; Joshi, A., S. Pingand K. J. Himmelstein, Patent Application WO 91/19481). Significant oralbioavailability is also achievable.

The peptide and protein modifications described herein mitigate and, insome cases, may eliminate the need for organic solvents. Trehalose,lactose, and mannitol and other saccharides have been used to preventaggregation. Aggregation of an anti-IgE humanized monoclonal antibodywas minimized by formulation with trehalose at or above a molar ratio inthe range of 300:1 to 500:1 (excipient:protein). However, the powderswere excessively cohesive and unsuitable for aerosol administration orexhibited unwanted protein glycation during storage (Andya, J. D., etal. (1999) Pharm Res 16: 350-358). Each of the additives discovered havelimitations as additives to therapeutics including xenobioticmetabolism, irritation or toxicity, or high cost. Contemplated for usewith the covalently modified peptides and/or proteins described hereinare excipients that are effective, non-irritating and non-toxic, do notrequire xenobiotic metabolism since they are comprised of the naturalsugars, fatty acids, or long chain alcohols, and which may also be usedto minimize aggregation in aqueous solutions or upon aqueousreconstitution of dried peptide and/or protein formulations in situ byphysiologic aqueous reconstitution by aqueous body fluids such as plasmaor saliva.

Other formulation components could include buffers and physiologicalsalts, non-toxic protease inhibitors such as aprotinin and soybeantrypsin inhibitor, alpha-1-antitrypsin, and protease-inactivatingmonoclonal antibodies, among others. Buffers could include organics suchas acetate, citrate, gluconate, fumarate, malate, polylysine,polyglutamate, chitosan, dextran sulfate, etc. or inorganics such asphosphate, and sulfate. Such formulations may additionally contain smallconcentrations of bacteriostatic agents like benzyl alcohol, and thelike.

Formulations suitable for intranasal administration also comprisesolutions or suspensions of the modified peptides and/or proteinproducts described herein in an acceptable evaporating solvents such ashydrofluoroalkanes. Such formulations are suitable for administrationfrom metered dose inhalers (MDI) and have advantages of lack of movementfrom site of administration, low irritation and absence of need forsterilization. Such formulations may also contain acceptable excipientsor bulking agents such as submicron anhydrous α-lactose.

In yet another aspect, the covalently modified peptides and/or proteinsdescribed herein exhibit increased shelf-life. As used herein, thephrase “shelf life” is broadly described as the length of time a productmay be stored without becoming unsuitable for use or consumption. The“shelf life” of the composition described herein, can also indicate thelength of time that corresponds to a tolerable loss in quality of thecomposition. The compositional shelf life as used herein isdistinguished from an expiration date; “shelf life” relates to thequality of the composition described herein, whereas “expiration date”relates more to manufacturing and testing requirements of thecomposition. For example, a composition that has passed its “expirationdate” may still be safe and effective, but optimal quality is no longerguaranteed by the manufacturer.

In some embodiments, the modified peptides described herein have greaterbioavailbility comparted to the unmodified peptide. In some embodiments,the bioavailability is greater for the modified peptides compared to theunmodified peptide when administered by nasal routes. In someembodiments, the bioavailability is greater for the modified peptidescompared to the unmodified peptide when administered by buccal. In someembodiments, the bioavailability is greater for the modified peptidescompared to the unmodified peptide when administered by oral routes. Insome embodiments, the bioavailability is greater for the modifiedpeptides compared to the unmodified peptide when administered by vaginalroutes. In some embodiments, the bioavailability is greater for themodified peptides compared to the unmodified peptide when administeredby inhalation routes. In some embodiments, the bioavailability isgreater for the modified peptides compared to the unmodified peptidewhen administered by intravenous routes. In some embodiments, thebioavailability is greater for the modified peptides compared to theunmodified peptide when administered by subcutaneous routes. In someembodiments, the bioavailability of the modified pepetides describedherein have between 10-90%, 20-80%, 30-60%, 10-30%, 15-30%, 15-45%,50-75%, 60-75%, 70-90%, 80-100%, 100-1000%, 100-500%, 50-200%, or100-200% greater biovavailability when compared with the unmodifiedpeptide. In some embodiments, the bioavailability of the modifiedpepetides described herein have at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 100%, at least 120%, at least 140%, at least 160%, atleast 180%, at least 200%, at least 220%, at least 240%, at least 260%,at least 280%, at least 300%, at least 400%, at least 500%, at least600%, at least 700%, at least 800%, at least 900%, at least 1000%, about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, about100%, about 120%, about 140%, about 160%, about 180%, about 200%, about220%, about 240%, about 260%, about 280%, about 300%, about 400%, about500%, about 600%, about 700%, about 800%, about 900% or about 1000%greater biovavailability when compared with the unmodified peptide.

Methods

In one aspect, provided herein are methods of administering to a subjectin need thereof an effective amount of the therapeutic compositionsdescribed herein. As used herein, “therapeutically effective amount” isinterchangeable with “effective amount” for purposes herein, and isdetermined by such considerations as are known in the art. The amountmust be effective to achieve a desired drug-mediated effect in thetreated subjects suffering from the disease thereof. A therapeuticallyeffective amount also includes, but is not limited to, appropriatemeasures selected by those skilled in the art, for example, improvedsurvival rate, more rapid recovery, or amelioration, improvement orelimination of symptoms, or other acceptable biomarkers or surrogatemarkers.

The compositions described herein are delivered to a vertebrate subjectin need of treatment including but not limited to, for example, a human.Moreover, depending on the condition being treated, these therapeuticcompositions may be formulated and administered systemically or locally.Techniques for formulation and administration may be found in the latestedition of “Remington's Pharmaceutical Sciences” (Mack Publishing Co,Easton Pa.). Suitable routes may, for example, include oral ortransmucosal administration; such as intranasal; buccal; ocular,vaginal; rectal; as well as parenteral delivery, includingintramuscular, subcutaneous, intravenous, or intraperitonealadministration.

In some embodiments of the methods, the effective amount of the peptideproduct for administration is from about 0.1 μg/kg/day to about 100.0μg/kg/day, or from 0.01 μg/kg/day to about 1 mg/kg/day or from 0.1μg/kg/day to about 50 mg/kg/day. In some embodiments, the peptideproduct is administered parenterally. In some embodiments, the peptideproduct is administered subcutaneously. In some embodiments, the methodof administration of the peptide product is nasal insufflation.

It will be understood, however, that the specific dose level andfrequency of dosage for any particular subject in need of treatment maybe varied and will depend upon a variety of factors including theactivity of the specific compound employed, the metabolic stability andduration of action of that compound, the age, body weight, generalhealth, sex, diet, mode and time of administration, rate of excretion,drug combination, the severity of the particular condition, and the hostundergoing therapy.

In one embodiment, provided is a method for chemically modifying amolecule by covalent linkage to a surfactant to increase or sustain thebiological action of the composition or molecule, for example, receptorbinding or enzymatic activity. In some embodiments, the molecule is apeptide. The method additionally can include further modificationcomprising covalent attachment of the molecule in the composition to apolymer such as polyethylene glycol.

The method(s) includes all aspects of the compositions described hereinincluding but not limited to compositions which reduce or eliminateimmunogenicity of peptide and/or protein drugs, are non-irritating, haveanti-bacterial or anti-fungal activity, have increased stability orbioavailability of a drug, decrease the bioavailability variance of thatdrug, avoid first pass liver clearance and reduce or eliminate anyadverse effects. As used herein, the term “immunogenicity” is theability of a particular substance or composition or agent to provoke animmunological response. The immunogenicity of the covalently modifiedpeptides and/or proteins described herein is confirmed by methods knownin the art.

Also provided is a method of administering a drug composition comprisinga peptide covalently linked to at least one alkyl glycoside anddelivered to a vertebrate, wherein the alkyl has from 1 to 30 carbonatoms, or further in the range of 6 to 16 carbon atoms, and the alkylglycoside increases the stability, bioavailability and/or duration ofaction of the drug.

In another embodiment, provided is a method of reducing or eliminatingimmunogenicity of a peptide and/or protein drug by covalently linkingthe peptide chain to at least one alkyl glycoside wherein the alkyl hasfrom 1 to 30 carbon atoms.

Throughout this application, various publications are referenced. Oneskilled in the art will understand that the referenced disclosures ofthese publications are hereby incorporated by reference into thisapplication.

Methods of Treatment

Provided herein, in some embodiments are methods for treatment of pain,including post-operative or chronic pain, comprising administration ofof a surfactant-modified peptide and/or protein product described herein(e.g., a peptide product of Formula I, II or III) to individuals in needthereof. In some of such embodiments, the opioid analogs describedherein are not addictive or habit forming, and/or are administered inlower dosages compared to current medications (e.g., codeine) and arelonger lasting compared to current medications.

Provided herein, in some embodiments are methods for prevention and/ortreatment of conditions associated with hypoparathyroidism and/ordecreases in bone mass density comprising administration of atherapeutically effective amount of a surfactant-modified peptide and/orprotein product described herein (e.g., a peptide product of Formula2-I-A, 2-III, 2-V, 2-VI or 2-VII) to individuals in need thereof. Insome embodiments, the conditions characterized by decreases in bone massdensity include, and are not limited to, osteoporosis, osteopenia,post-menopausal osteoporosis, Paget's disease, glucocorticoid inducedosteoporosis, old age osteoporosis, humoral hypercalcemia, or the like.

In some embodiments, provided herein are methods for treatment ofhypoparathyroidism comprising administration of a therapeuticallyeffective amount of a surfactant-modified peptide and/or protein productdescribed herein (e.g., a peptide product of Formula 2-I-A, 2-III, 2-V,2-VI or 2-VII) to individuals in need thereof. In some embodiments, thehypoparathyroidism is associated with decrease in bone mass density.

Further provided herein are methods for stimulating bone repair and/orfavoring engraftment of a bone implant comprising administration of atherapeutically effective amount of a surfactant-modified peptide and/orprotein product described herein (e.g., a peptide product of Formula2-I-A, 2-III, 2-V, 2-VI or 2-VII) to individuals in need thereof.

In yet further embodiments, provided herein are methods for increasingbone density and/or reducing incidence of fractures (e.g., vertebraefractures, hip fractures, or the like) comprising administration of atherapeutically effective amount of a surfactant-modified peptide and/orprotein product described herein (e.g., a peptide product of Formula2-I-A, 2-III, 2-V, 2-VI or 2-VII) to individuals in need thereof.

In some embodiments, provided herein are methods for treatment ofhumoral hypercalcemia comprising administration of a therapeuticallyeffective amount of a surfactant-modified peptide and/or protein productdescribed herein (e.g., a peptide product of Formula 2-I-A, 2-III, 2-V,2-VI or 2-VII) to individuals in need thereof. In some embodiments,humoral hypercalcemia is associated with tumors. In some of suchembodiments, the peptide product (e.g., a peptide product of Formula2-I-A, 2-III, 2-V, 2-VI or 2-VII) is an inverse agonist or an antagonistof PTH or PTHrP.

In some embodiments of the methods described above, the peptide and/orprotein that is covalently attached to a surfactant is PTH, PTHrP, or ananalog thereof. In some embodiments, the surfactant-modified peptideand/or protein (e.g., a peptide product of Formula 2-I-A, 2-III, 2-V,2-VI or 2-VII) is administered prophylactically and delays occurrence ofany condition associated with loss of bone density, including and notlimited to osteoporosis, osteopenia, post-menopausal osteoporosis,Paget's disease, glucocorticoid induced osteoporosis, old ageosteoporosis, or the like. In some embodiments, the surfactant-modifiedpeptide and/or protein (e.g., a peptide product of Formula 2-I-A, 2-III,2-V, 2-VI or 2-VII) is administered therapeutically and delaysprogression of any condition associated with loss of bone density,including and not limited to osteoporosis, osteopenia, post-menopausalosteoporosis, Paget's disease, glucocorticoid induced osteoporosis,humoral hypercalcemia, or the like. In some embodiments, thesurfactant-modified peptide and/or protein (e.g., a peptide product ofFormula 2-I-A, 2-III, 2-V, 2-VI or 2-VII) is administeredprophylactically and/or therapeutically and delays progression ofosteopenia to osteoporosis. In some embodiments, the surfactant-modifiedpeptide and/or protein (e.g., a peptide product of Formula 2-I-A, 2-III,2-V, 2-VI or 2-VII) is administered prophylactically and/ortherapeutically and reduces or halts further loss of bone density,thereby stabilizing disease.

In some embodiments, the surfactant-modified peptide and/or protein(e.g., a peptide product of Formula 2-I-A, 2-III, 2-V, 2-VI or 2-VII) isadministered parenterally. In some embodiments, the surfactant-modifiedpeptide and/or protein (e.g., a peptide product of Formula 2-I-A, 2-III,2-V, 2-VI or 2-VII) is administered subcutaneously. In some embodiments,the surfactant-modified peptide and/or protein (e.g., a peptide productof Formula 2-I-A, 2-III, 2-V, 2-VI or 2-VII) is administered by nasalinsufflation.

In some embodiments of the methods described above, thesurfactant-modified peptide and/or protein (e.g., a peptide product ofFormula 2-I-A, 2-III, 2-V, 2-VI or 2-VII) has a longer duration ofaction compared to a pharmaceutical comprising currently knowntherapeutics (e.g., recombinant PTH, bisphosphonates, antibodyDenosumab, or the like). In some embodiments, the surfactant-modifiedpeptide and/or protein (e.g., a peptide product of Formula 2-I-A, 2-III,2-V, 2-VI or 2-VII) is administered for longer period of time(e.g., >two years) compared to a pharmaceutical comprising currentlyknown therapeutics (e.g., recombinant PTH, bisphosphonates, antibodyDenosumab, or the like) while reducing or ameliorating side-effects(e.g., osteonecrosis in jaw, skin infections or the like) associatedwith currently known therapeutics (e.g., recombinant PTH,bisphosphonates, antibody Denosumab, or the like). In some embodimentsof the methods described above, the surfactant-modified peptide and/orprotein (e.g., a peptide product of Formula 2-I-A, 2-III, 2-V, 2-VI or2-VII) is an agonist of PTH or PTHrP. In some embodiments of the methodsdescribed above, the surfactant-modified peptide and/or protein (e.g., apeptide product of Formula 2-I-A, 2-III, 2-V, 2-VI or 2-VII) is anantagonist of PTH or PTHrP. In some embodiments of the methods describedabove, the surfactant-modified peptide and/or protein (e.g., a peptideproduct of Formula 2-I-A, 2-III, 2-V, 2-VI or 2-VII) is an inverseagonist of PTH or PTHrP.

Provided herein, in some embodiments are methods for prevention and/ortreatment of conditions associated with decreases in insulin sensitivitycomprising administration of a therapeutically effective amount of asurfactant-modified peptide and/or protein product described herein(e.g., a peptide product of Formula 3-I-A, 3-III-A, 3-III-B or Formula3-V) to individuals in need thereof. In some embodiments, the conditionscharacterized by decreases in insulin sensitivity include, and are notlimited to, the metabolic syndrome, obesity-related insulin resistance,hypertension, systemic inflammation associated with high C reactiveprotein, diabetes, or the like.

Also provided is a method of treating conditions associated with insulinresistance including and not limited to obesity, the metabolic syndrome,type 2 diabetes, hypertension, atherosclerosis or the like, comprisingadministering a drug composition comprising a peptide covalently linkedto at least one alkyl glycoside and delivered to a vertebrate, whereinthe alkyl has from 1 to 30 carbon atoms, or further in the range of 6 to18 carbon atoms (e.g., a peptide product of Formula 3-I-A, 3-III-A,3-III-B or Formula 3-V), and wherein covalent linkage of the alkylglycoside to the peptide increases the stability, bioavailability and/orduration of action of the drug.

Also provided herein are methods for treatment of insulin resistancecomprising administration of a therapeutically effective amount of asurfactant-modified peptide and/or protein product described herein(e.g., a peptide product of Formula 3-I-A, 3-III-A, 3-III-B or Formula3-V) to individuals in need thereof. In some embodiments, the insulinresistance is associated with the metabolic syndrome (Syndrome X) and/ordiabetes.

Further provided herein are methods for stimulating resensitization ofthe body to insulin comprising administration of a therapeuticallyeffective amount of a surfactant-modified peptide and/or protein productdescribed herein (e.g. a peptide product of Formula 3-I-A, 3-III-A,3-III-B or Formula 3-V) to individuals in need thereof.

In yet further embodiments, provided herein are methods for increasinginsulin sensitivity through weight loss, comprising administration of atherapeutically effective amount of a surfactant-modified peptide and/orprotein product described herein (e.g. a peptide product of Formula3-I-A, 3-III-A, 3-III-B or Formula 3-V and in Table 1 of FIG. 1 andTable 2 of FIG. 2) to individuals in need thereof.

Also provided herein are methods of treating diabetes or prediabetescomprising administering to a subject in need thereof a therapeuticallyeffective amount of a peptide product described above and herein and inTable 1 of FIG. 1 and Table 2 of FIG. 2 to an individual in needthereof.

Provided herein are methods for treating or delaying the progression oronset of conditions selected from diabetes, diabetic retinopathy,diabetic neuropathy, diabetic nephropathy, insulin resistance,hyperglycemia, hyperinsulinemia, metabolic syndrome, diabeticcomplications, elevated blood levels of free fatty acids or glycerol,hyperlipidemia, obesity, hypertriglyceridemia, atherosclerosis, acutecardiovascular syndrome, infarction, ischemic reperfusion ahypertension, comprising administering a therapeutically effectiveamount of a peptide product described herein and in Table 1 of FIG. 1and Table 2 of FIG. 2 to an individual in need thereof. In an additionalembodiment, provided herein are methods for treating delays in woundhealing comprising administering a therapeutically effective amount of apeptide product described herein and in Table 1 of FIG. 1 and Table 2 ofFIG. 2 to an individual in need thereof.

In one embodiment said condition to be treated is diabetes. In oneembodiment said condition to be treated is insulin resistance. In oneembodiment said condition to be treated is the metabolic syndrome. Inone embodiment said effective amount of said peptide is from about 0.1rig/kg/day to about 100.0 rig/kg/day.

In one embodiment the method of administration is parenteral. In oneembodiment the method of administration is per oral. In one embodimentthe method of administration is subcutaneous. In one embodiment themethod of administration is nasal insufflation.

Further provided herein is a method of reducing weight gain or inducingweight loss comprising administering a therapeutically effective amountof a peptide product described herein and in Table 1 of FIG. 1 and Table2 of FIG. 2 to an individual in need thereof. In some embodiments, theweight gain is associated with metabolic syndrome.

Provided herein is a method of treating hypoglycemia comprisingadministering a therapeutically effective amount of a peptide productdescribed herein and in Table 1 of FIG. 1 and Table 2 of FIG. 2 to anindividual in need thereof.

Also provided herein are methods for treatment of diabetes comprisingadministering a therapeutically effective amount of a peptide productdescribed herein and in Table 1 of FIG. 1 and Table 2 of FIG. 2 to anindividual in need thereof and at least one additional therapeuticagent; wherein said therapeutic agent is selected from an antidiabeticagent, an anti-obesity agent, a satiety agent, an anti-inflammatoryagent, an anti-hypertensive agent, an anti-atherosclerotic agent and alipid-lowering agent.

In some embodiments of the methods described above, the peptide and/orprotein that is covalently attached to a surfactant is a glucagon orGLP-1 peptide, or an analog thereof. In some embodiments, thesurfactant-modified peptide and/or protein (e.g., a peptide product ofFormula 3-I-A, 3-III-A, 3-III-B or Formula 3-V) is administeredprophylactically and delays occurrence of any condition associated withinsulin resistance, including and not limited to the metabolic syndrome,hypertension, diabetes, type 2 diabetes, gestational diabetes,hyperlipidemia, atherosclerosis, systemic inflammation or the like. Insome embodiments, the surfactant-modified peptide and/or protein (e.g.,a peptide product of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V) isadministered therapeutically and delays progression of any conditionassociated with the metabolic syndrome, hypertension, diabetes, type 2diabetes, gestational diabetes, hyperlipidemia, atherosclerosis,systemic inflammation or the like. In some embodiments, thesurfactant-modified peptide and/or protein (e.g., a peptide product ofFormula 3-I-A, 3-III-A, 3-III-B or Formula 3-V) is administeredprophylactically and/or therapeutically and delays progression ofinsulin resistance to diabetes. In some embodiments, thesurfactant-modified peptide and/or protein (e.g., a peptide product ofFormula 3-I-A, 3-III-A, 3-III-B or Formula 3-V) is administeredprophylactically and/or therapeutically and reduces or halts furtherloss of insulin resistance, thereby stabilizing disease.

In some embodiments, the surfactant-modified peptide and/or protein(e.g., a peptide product of Formula 3-I-A, 3-III-A, 3-III-B or Formula3-V) is administered parenterally. In some embodiments, thesurfactant-modified peptide and/or protein (e.g., a peptide product ofFormula 3-I-A, 3-III-A, 3-III-B or Formula 3-V) is administeredsubcutaneously. In some embodiments, the surfactant-modified peptideand/or protein (e.g., a peptide product of Formula 3-I-A, 3-III-A,3-III-B or Formula 3-V) is administered by nasal insufflation.

In some embodiments of the methods described above, thesurfactant-modified peptide and/or protein (e.g., a peptide product ofFormula 3-I-A, 3-III-A, 3-III-B or Formula 3-V) has a longer duration ofaction compared to a pharmaceutical comprising currently knowntherapeutics (e.g., exenatide, metformin or the like).

Combination Therapy with PTH Analogs

Is some embodiments of the methods described above, thesurfactant-modified peptide and/or protein (e.g., a peptide product ofFormula 2-I-A, 2-III, 2-V, 2-VI or 2-VII) is administered in combinationwith a bone resorption inhibitor including, a bisphosphonate, (e.g.alendronate) or strontium salt; or a substance with estrogen-likeeffect, e.g. estrogen; or a selective estrogen receptor modulator, e.g.raloxifene, tamoxifene, droloxifene, toremifene, idoxifene, orlevormeloxifene; or a calcitonin-like substance, e.g. calcitonin; or avitamin D analog; or a calcium salt. The therapeutic agents areoptionally administered simultaneously, or sequentially in any order. Byway of example, in some embodiments of the methods described above, afirst regimen of the surfactant-modified peptide and/or protein (e.g., apeptide product of Formula 2-I-A, 2-III, 2-V, 2-VI or 2-VII) isadministered to an individual in need thereof, and the regimen isfollowed by a second regimen of bisphosphonate therapy. By way ofexample, in some other embodiments, a first regimen of thesurfactant-modified peptide and/or protein (e.g., a peptide product ofFormula 2-I-A, 2-III, 2-V, 2-VI or 2-VII) is administered to anindividual in need thereof, followed by a drug holiday, followed by asecond regimen of estrogen receptor modulators.

Combination Therapy with GLP Analogs

In some embodiments of the methods described above, thesurfactant-modified peptide and/or protein (e.g., a peptide product ofFormula 3-I-A, 3-III-A, 3-III-B or Formula 3-V) is administered incombination with other methods of treatment of the metabolic syndromeselected from the group comprising an antidiabetic agent, ananti-obesity agent, an anti-hypertensive agent, an anti-atheroscleroticagent and a lipid-lowering agent. By way of example, efficaciousantidiabetic agents suitable for administration in combination with asurfactant-modified peptide and/or protein product described hereininclude a biguanide, a sulfonylurea, a glucosidase inhibitor a PPAR γagonist, a PPAR α/γ dual agonist, an aP2 inhibitor, a DPP4 inhibitor, aninsulin sensitizer, a GLP-1 analog, insulin and a meglitinide.Additional examples include metformin, glyburide, glimepiride,glipyride, glipizide, chlorpropamide, gliclazide, acarbose, miglitol,pioglitazone, troglitazone, rosiglitazone, muraglitazar, insulin,G1-262570, isaglitazone, JTT-501, NN-2344, L895 645, YM-440, R-119702,A19677, repaglinide, nateglinide, KAD 1129, AR-HO 39242, GW-40 I 5 44,KRP2 I 7, AC2993, LY3 I 5902, NVP-DPP-728A and saxagliptin.

In some embodiments of the methods described above, thesurfactant-modified peptide and/or protein (e.g., a peptide product ofFormula 3-I-A, 3-III-A, 3-III-B or Formula 3-V) is administered incombination with other methods of treatment of the metabolic syndromeselected from the group of efficacious anti-obesity agents. By way ofexample, efficacious anti-obesity agents suitable for administrationwith the peptide products described herein include beta 3 adrenergicagonist, a lipase inhibitor, a serotonin (and dopamine) reuptakeinhibitor, a thyroid receptor beta compound, a CB-1 antagonist, a NPY-Y2and a NPY-Y4 receptor agonist and an anorectic agent. Specific membersof these classes comprise orlistat, AfL-962, A1967l, L750355, CP331648,sibutramine, topiramate, axokine, dexamphetamine, phentermine,phenylpropanolamine, rimonabant (SR1 4I7164), and mazindol.

In some embodiments of the methods described above, thesurfactant-modified peptide and/or protein (e.g., a peptide product ofFormula 3-I-A, 3-III-A, 3-III-B or Formula 3-V) is administered incombination with other methods of treatment of the metabolic syndromeselected from the group of efficacious lipid-lowering agents. By way ofexample, efficacious lipid-lowering agents suitable for administrationwith the peptide products described herein include agents selected fromthe group consisting of an MTP inhibitor, cholesterol ester transferprotein, an HMG CoA reductase inhibitor, a squalene synthetaseinhibitor, a fibric acid derivative, an upregulator of LDL receptoractivity, a lipoxygenase inhibitor, and an ACAT inhibitor. Specificexamples from these classes comprise pravastatin, lovastatin,simvastatin, atorvastatin, cerivastatin, fluvastatin, nisvastatin,visastatin, fenofibrate, gemfibrozil, clofibrate, avasimibe, TS-962,MD-700, CP-52941 4, and LY295 427.

In some embodiments of the methods described above, thesurfactant-modified peptide and/or protein (e.g., a peptide product ofFormula 3-I-A, 3-III-A, 3-III-B or Formula 3-V) is administered incombination with peptide hormones, and analogs thereof, that are knownto exhibit pro-satiety effects in animal models and in man. Contemplatedwithin the scope of embodiments presented herein is a combination of thepeptide products described herein and long-acting satiety agents fortreatment of obesity. Examples of such peptide satiety agents includeGLP-1, pancreatic polypeptide (PP), cholecystokinin (CCK), peptide YY(PYY), amylin, calcitonin, OXM, neuropeptide Y (NPY), and analogsthereof (Bloom, S. R., et al. (2008) Mol Interv 8: 82-98; Field, B. C.,et al. (2009) Br J Clin Pharmacol 68: 830-843).

Also contemplated within the scope of embodiments presented herein aremethods for treatment of obesity comprising administration of peptideproducts described herein in combination with peptide hormones includingand not limited to leptin, ghrelin and CART (cocaine- andamphetamine-regulated transcript) analogs and antagonists.

Additional peptide products in the body are associated with fat cells orthe obese state (adipokines) and are known to have proinflammatoryeffects (Gonzalez-Periz, A. and Claria, J. (2010) ScientificWorldJournal10: 832-856). Such agents will have additional favorable actions whenused in combination with the peptide products described herein. Examplesof agents that offer a beneficial effect when used in combination withthe peptide products described herein include analogs and antagonists ofadiponectin, chemerin, visfatin, nesfatin, omentin, resistin, TNFalpha,IL-6 and obestatin.

Dosing

The covalently modified peptides and/or proteins described herein may beadministered in any amount to impart beneficial therapeutic effect in anumber of disease states. In some embodiments, covalently modifiedpeptides and/or proteins described herein are useful in the treatment ofinflammation. In an embodiment, compounds presented herein impartbeneficial activity in the modulation of post-operative or chronic pain.In an embodiment, the present peptides are administered to a patient atconcentrations higher or lower than that of other forms of treatmentwhich modulate pain. In yet another embodiment, the present peptides areadministered with other compounds to produce synergistic therapeuticeffects.

Representative delivery regimens include oral, parenteral (includingsubcutaneous, intramuscular and intravenous injection), rectal, buccal(including sublingual), transdermal, inhalation ocular and intranasal.An attractive and widely used method for delivery of peptides entailssubcutaneous injection of a controlled-release injectable formulation.In some embodiments, covalently modified peptides and/or proteinsdescribed herein are useful for subcutaneous, intranasal and inhalationadministration.

The selection of the exact dose and composition and the most appropriatedelivery regimen will be influenced by, inter alia, the pharmacologicalproperties of the selected peptide, the nature and severity of thecondition being treated, and the physical condition and mental acuity ofthe recipient. Additionally, the route of administration will result indifferential amounts of absorbed material. Bioavailabilities foradministration of peptides through different routes are particularlyvariable, with amounts from less than 1% to near 100% being seen.Typically, bioavailability from routes other than intravenous,intraperitoneal or subcutaneous injection are 50% or less.

In general, covalently modified peptides and/or proteins describedherein, or salts thereof, are administered in amounts between about0.001 and 20 mg/kg body weight per day, between about 0.01 and 10 mg/kgbody weight per day, between about 0.1 and 1000 g/kg body weight perday, or between about 0.1 to about 100 g/kg body weight per day. Routesof administration vary. For example, covalently modified opiod peptidesand/or proteins described herein, or salts thereof, are administered inamounts between about 0.1 and 1000 g/kg body weight per day, or betweenabout 0.1 to about 100 g/kg body weight per day, by subcutaneousinjection. By way of example, for a 50 kg human female subject, thedaily dose of active ingredient is from about 5 to about 5000 μg, orfrom about 5 to about 5000 μg by subcutaneous injection. Different doseswill be needed, depending on the route of administration, the compoundpotency, the pharmacokinetic profile and the applicable bioavailabilityobserved, and the active agent and the disease being treated. In analternate embodiment where the administration is by inhalation, thedaily dose is from 1000 to about 20,000 μg, twice daily. In othermammals, such as horses, dogs, and cattle, higher doses may be required.This dosage may be delivered in a conventional pharmaceuticalcomposition by a single administration, by multiple applications, or viacontrolled release, as needed to achieve the most effective results.

Pharmaceutically acceptable salts retain the desired biological activityof the parent peptide without toxic side effects. Examples of such saltsare (a) acid addition salts formed with inorganic acids, for examplehydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid,nitric acid and the like; and salts formed with organic acids such as,for example, acetic acid, trifluoroacetic acid, tartaric acid, succinicacid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid,ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid,polyglutamic acid, naphthalenesulfonic acids, naphthalene disulfonicacids, polygalacturonic acid and the like; (b) base addition salts orcomplexes formed with polyvalent metal cations such as zinc, calcium,bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium,and the like; or with an organic cation formed fromN,N′-dibenzylethylenediamine or ethylenediamine; or (c) combinations of(a) and (b), e.g., a zinc tannate salt and the like.

Also contemplated, in some embodiments, are pharmaceutical compositionscomprising as an active ingredient covalently modified peptides and/orproteins described herein, or pharmaceutically acceptable salt thereof,in admixture with a pharmaceutically acceptable, non-toxic carrier. Asmentioned above, such compositions may be prepared for parenteral(subcutaneous, intramuscular or intravenous) administration,particularly in the form of liquid solutions or suspensions; for oral orbuccal administration, particularly in the form of tablets or capsules;for intranasal administration, particularly in the form of powders,nasal drops, evaporating solutions or aerosols; for inhalation,particularly in the form of liquid solutions or dry powders withexcipients, defined broadly; and for rectal or transdermaladministration.

The compositions may conveniently be administered in unit dosage formand may be prepared by any of the methods well-known in thepharmaceutical art, for example as described in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,(1985), incorporated herein by reference. Formulations for parenteraladministration may contain as excipients sterile water or saline,alkylene glycols such as propylene glycol, polyalkylene glycols such aspolyethylene glycol, saccharides, oils of vegetable origin, hydrogenatednaphthalenes, serum albumin nanoparticles (as used in Abraxane™,American Pharmaceutical Partners, Inc. Schaumburg Ill.), and the like.For oral administration, the formulation can be enhanced by the additionof bile salts or acylcarnitines. Formulations for nasal administrationmay be solid or solutions in evaporating solvents such ashydrofluorocarbons, and may contain excipients for stabilization, forexample, saccharides, surfactants, submicron anhydrous α-lactose ordextran, or may be aqueous or oily solutions for use in the form ofnasal drops or metered spray. For buccal administration typicalexcipients include sugars, calcium stearate, magnesium stearate,pregelatinated starch, and the like.

When formulated for nasal administration, the absorption across thenasal mucous membrane may be further enhanced by surfactants, such asfor example, glycocholic acid, cholic acid, taurocholic acid, ethocholicacid, deoxycholic acid, chenodeoxycholic acid, dehydrocholic acid,glycodeoxycholic acid, cyclodextrins and the like in an amount in therange between about 0.1 and 15 weight percent, between about 0.5 and 4weight percent, or about 2 weight percent. An additional class ofabsorption enhancers reported to exhibit greater efficacy with decreasedirritation is the class of alkyl maltosides, such as tetradecylmaltoside(Arnold, J. J., et al. (2004) J Pharm Sci 93: 2205-2213, Ahsan, F., etal. (2001) Pharm Res 18: 1742-1746) and references therein, all of whichare hereby incorporated by reference.

When formulated for delivery by inhalation, a number of formulationsoffer advantages. Adsorption of the active peptide to readily dispersedsolids such as diketopiperazines (for example Technosphere particles;(Pfutzner, A. and Forst, T. (2005) Expert Opin Drug Deliv 2: 1097-1106)or similar structures gives a formulation which results in a rapidinitial uptake of the therapeutic agent. Lyophylized powders, especiallyglassy particles, containing the active peptide and an excipient areuseful for delivery to the lung with good bioavailability, for example,see Exubera® (inhaled insulin by Pfizer and Aventis PharmaceuticalsInc.). Additional systems for delivery of peptides by inhalation aredescribed (Mandal, T. K., Am. J. Health Syst. Pharm. 62: 1359-64(2005)).

Delivery of covalently modified peptides and/or proteins describedherein to a subject over prolonged periods of time, for example, forperiods of one week to one year, may be accomplished by a singleadministration of a controlled release system containing sufficientactive ingredient for the desired release period. Various controlledrelease systems, such as monolithic or reservoir-type microcapsules,depot implants, polymeric hydrogels, osmotic pumps, vesicles, micelles,liposomes, transdermal patches, iontophoretic devices and alternativeinjectable dosage forms may be utilized for this purpose. Localizationat the site to which delivery of the active ingredient is desired is anadditional feature of some controlled release devices, which may provebeneficial in the treatment of certain disorders.

One form of controlled release formulation contains the peptide or itssalt dispersed or encapsulated in a slowly degrading, non-toxic,non-antigenic polymer such as copoly(lactic/glycolic) acid, as describedin the pioneering work of Kent, Lewis, Sanders, and Tice, U.S. Pat. No.4,675,189, incorporated by reference herein. The compounds, or theirsalts, may also be formulated in cholesterol or other lipid matrixpellets, or silastomer matrix implants. Additional slow release, depotimplant or injectable formulations will be apparent to the skilledartisan. See, for example, Sustained and Controlled Release DrugDelivery Systems, J. R. Robinson ed., Marcel Dekker, Inc., New York,1978, and R. W. Baker, Controlled Release of Biologically Active Agents,John Wiley & Sons, New York, 1987.

An additional form of controlled-release formulation comprises asolution of a biodegradable polymer, such as copoly(lactic/glycolicacid) or block copolymers of lactic acid and PEG, is bioacceptablesolvent, which is injected subcutaneously or intramuscularly to achievea depot formulation. Mixing of the peptides described herein with such apolymeric formulation is suitable to achieve very long duration ofaction formulations.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindependent publication or patent application is specifically andindividually indicated to be incorporated by reference.

The covalently modified peptides and/or proteins described herein andthe reagents for the synthesis thereof are more particularly describedin the following examples which are intended as illustrative only sincenumerous modifications and variations therein will be apparent to thoseof ordinary skill in the art.

Examples Example 1: Reagents—N-α-Fmoc, N-ε-(1-octylβ-D-glucuronide-6-yl)-L-lysine

In an oven-dried 250 mL Erlenmeyer flask is placed 1-octylβ-D-glucuronic acid (Carbosynth Ltd., 3.06 g, 10 mmol), 50 mL anhydrousDMF, and anhydrous HOBT (1.62 g, 12 mmol). A chilled (4° C.) solution ofDCC (2.48 g, 12 mmol) in 50 mL of DMF is added, with stirring, and thereaction is allowed to proceed for 5 min. The copious white precipitateof N, N′-dicyclohexylurea is filtered on a fritted glass funnel and thefiltrate is added to a solution of N-α-Fmoc-L-lysine (3.68 g, 10 mmol)in 25 ml anhydrous DMF. The reaction is allowed to proceed for 25 minwith warming to room temp or until the ninhydrin color is very faint.The reaction mixture is filtered, stripped to dryness and crystallizedfrom MeOH/Et₂O by dissolution in MeOH and slow dilution to the cloudpoint with Et₂O, followed by refrigeration. Further purification can beachieved by silica gel chromatography using a solvent gradient fromEtOAc to EtOAc/EtOH/AcOH.

In a similar manner, but substituting N-α-Boc-L-lysine is obtainedN-α-Boc,N-ε-(1-octyl β-D-glucuronide-6-yl)-L-lysine, suitable forN-terminal incorporation and cleavage to a free N-Terminus. In a similarmanner, but substituting N-α-Ac-L-lysine is obtained N-α-Ac,N-ε-(1-octylβ-D-glucuronide-6-yl)-L-lysine, suitable for incorporation at theN-terminus of a peptide with a blocked N-terminus. In a similar manner,but substituting the appropriate amount of N-α-Fmoc-L-ornithine isobtained N-α-Fmoc,N-δ-(1-octyl β-D-glucuronide-6-yl)-L-ornithine. In asimilar manner but substituting other N-mono-protected diamino acids oneobtains the corresponding reagents. Alternatively, use of a transientMe₃Si ester protecting group during the coupling and withoutpreactivation of the 1-octyl β-D-glucuronic acid provides a facile routeto the formation of the reagents. The transient Me₃Si ester is producedby reaction of the Fmoc-Lys-OH with an equimolar amount ofN,O-bis(trimethylsilyl)acetamide in dichloromethane (CH₂Cl₂). Theorganic layer contains the desired reagent as a solution in CH₂Cl₂ readyfor coupling with the 1-alkyl glucoronide as above. The filteredreaction mixture is washed with aqueous NaHSO₄ to hydrolyze the Me₃Siester, dried over MgSO₄ and solvent is removed.

Similarly, but using peracetyl or perbenzoyl 1-octyl β-D-glucuronic acidone obtains the Ac, or Bz protected form of the reagents (e.g.2,3,4-trisacetyl 1-octyl β-D-glucuronic acid, and the like, formed bytreatment with Ac₂O). Such reagents have increased stability during acidcleavage from the resin and are used when instability duringdeprotection is detected, see (Kihlberg, J., et al. (1997) MethodsEnzymol 289: 221-245) and references therein. Final deprotection of suchproducts is carried out by base-catalyzed transesterification aftercleavage, by use of MeOH/NH₃, MeOH/NaOMe, MeOH/NH₂NH₂, as describedabove.

Example 2: Synthetic Peptide Analogs

In general, peptide synthesis methods involve the sequential addition ofprotected amino acids to a growing peptide chain. Normally, either theamino or carboxyl group of the first amino acid and any reactive sidechain group are protected. This protected amino acid is then eitherattached to an inert solid support, or utilized in solution, and thenext amino acid in the sequence, also suitably protected, is added underconditions amenable to formation of the amide linkage. After all thedesired amino acids have been linked in the proper sequence, protectinggroups and any solid support are removed to afford the crude peptide.The peptide is desalted and purified chromatographically.

A preferred method of preparing the analogs of the physiologicallyactive truncated peptides, having fewer than about fifty amino acids,involves solid phase peptide synthesis. In this method the α-amino (Nα)functions and any reactive side chains are protected by acid- orbase-sensitive groups. The protecting group should be stable to theconditions of peptide linkage formation, while being readily removablewithout affecting the extant peptide chain. Suitable α-amino protectinggroups include, but are not limited to t-butoxycarbonyl (Boc),benzyloxycarbonyl (Cbz), o-chlorobenzyloxycarbonyl,biphenylisopropyloxycarbonyl, t-amyloxycarbonyl (Amoc),isobornyloxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxy-carbonyl,o-nitrophenylsulfenyl, 2-cyano-t-butoxycarbonyl,9-fluorenyl-methoxycarbonyl (Fmoc) and the like, preferably Boc or morepreferably, Fmoc. Suitable side chain protecting groups include, but arenot limited to: acetyl, benzyl (Bzl), benzyloxymethyl (Bom), Boc,t-butyl, o-bromobenzyloxycarbonyl, t-butyl, t-butyldimethylsilyl,2-chlorobenzyl (Cl-z), 2,6-dichlorobenzyl, cyclohexyl, cyclopentyl,isopropyl, pivalyl, tetrahydropyran-2-yl, tosyl (Tos),2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), trimethylsilyland trityl. A preferred Nα-protecting group for synthesis of thecompounds is the Fmoc group. Preferred side chain protecting groups areO-t-Butyl group for Glu, Tyr, Thr, Asp and Ser; Boc group for Lys andTrp side chains; Pbf group for Arg; Trt group for Asn, Gln, and His. Forselective modification of a Lys residue, orthogonal protection with aprotecting group not removed by reagents that cleave the Fmoc or t-butylbased protecting groups is preferred. Preferred examples formodification of the Lys side chain include, but are not limited to,those removed by hydrazine but not piperidine; for example1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde) or1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) andallyloxycarbonyl (Alloc).

The Fmoc-Lys(ivDde) or Fmoc-Lys(Dde) protecting group scheme ispreferred in cases where side chain lactam formation is desired(Houston, M. E., Jr., et al. (1995) J Pept Sci 1: 274-282; Murage, E.N., et al. (2010) J Med Chem), since in this case Fmoc-Glu(O-Allyl) andFmoc-Lys(Alloc) can be incorporated and used to provide transientprotection, then deprotected for lactam formation while the Lys(Dde)protecting group remains for later removal and reaction with thefunctionalized surfactant. The side chain lactam between acidic andbasic residue (e.g. Glu and Lys) is carried out after removal of theallyl-based protection by activation of the carboxyl side chain functionwith N,N′-diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) or2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate(HBTU)/N,N-di-isopropylethylamine (DIEA), using standard protocols wellknown to the art.

In solid phase synthesis, the C-terminal amino acid is first attached toa suitable resin support. Suitable resin supports are those materialswhich are inert to the reagents and reaction conditions of the stepwisecondensation and deprotection reactions, as well as being insoluble inthe media used. Examples of commercially available resins includestyrene/divinylbenzene resins modified with a reactive group, e.g.,chloromethylated co-poly-(styrene-divinylbenzene), hydroxymethylatedco-poly-(styrene-divinylbenzene), and the like. Benzylated,hydroxymethylated phenylacetamidomethyl (PAM) resin is preferred for thepreparation of peptide acids. When the C-terminus of the compound is anamide, preferred resins arep-methylbenzhydrylamino-co-poly(styrene-divinyl-benzene) resin, a 2,4dimethoxybenzhydrylamino-based resin (“Rink amide”),4-Hydroxymethylphenoxyacetyl aminomethyl resin (HMP Am) and the like. Anespecially preferred support for the synthesis of larger peptides arecommericially available resins containing PEG sequences grafted ontoother polymeric matricies, such as the Rink Amide-PEG and PAL-PEG-PSresins (Applied Biosystems) or similar resins designed for peptide amidesynthesis using the Fmoc protocol. Thus in certain cases it is desirableto have an amide linkage to a PEG chain. It those cases it is convenientto link an N-Fmoc-amino-PEG-carboxylic acid to the amide forming resinabove (e.g. Rink amide resin and the like). The first amino acid of thechain can be coupled as an N-Fmoc-amino acid to the amino function ofthe PEG chain. Final deprotection will yield the desiredPeptide-NH-PEG-CO—NH₂ product.

Attachment to the PAM resin may be accomplished by reaction of the Nαprotected amino acid, for example the Boc-amino acid, as its ammonium,cesium, triethylammonium, 1,5-diazabicyclo-[5.4.0]undec-5-ene,tetramethylammonium, or similar salt in ethanol, acetonitrile,N,N-dimethylformamide (DMF), and the like, preferably the cesium salt inDMF, with the resin at an elevated temperature, for example betweenabout 40° and 60° C., preferably about 50° C., for from about 12 to 72hours, preferably about 48 hours. This will eventually yield the peptideacid product following acid cleavage or an amide following aminolysis.

The Nα-Boc-amino acid may be attached to the benzhydrylamine or HMP Amresin by means of, for example, a DIC/HOBt mediated coupling for fromabout 2 to about 24 hours, preferably about 2 hours at a temperature ofbetween about 10° and 50° C., preferably 25° C. in a solvent such asCH₂Cl₂ or DMF, preferably CH₂Cl₂.

For Boc-based protocols, the successive coupling of protected aminoacids may be carried out by methods well known in the art, typically inan automated peptide synthesizer. Following neutralization withtriethylamine, DIEA, N-methylmorpholine (NMM), collidine, or similarbase, each protected amino acid is introduced in approximately about 1.5to 2.5 fold molar excess and the coupling carried out in an inert,nonaqueous, polar solvent such as CH₂Cl₂, DMF, N-methylpyrrolidone(NMP), N,N-dimethylacetamide (DMA), or mixtures thereof, preferably indichloromethane at ambient temperature. For Fmoc-based protocols no acidis used for deprotection but a base, preferably DIEA or NMM, is usuallyincorporated into the coupling mixture. Couplings are typically done inDMF, NMP, DMA or mixed solvents, preferably DMF. Representative couplingagents are N,N′-dicyclohexylcarbodiimide (DCC), DIC or othercarbodiimide, either alone or in the presence of HOBt, O-acyl ureas,benzotriazol-1-yl-oxytris(pyrrolidino)phosphonium hexafluorophosphate(PyBop), N-hydroxysuccinimide, other N-hydroxyimides, or oximes.Alternatively, protected amino acid active esters (e.g. p-nitrophenyl,pentafluorophenyl and the like) or symmetrical anhydrides may be used.Preferred coupling agents are of the aminium/uronium (alternativenomenclatures used by suppliers) class such as HBTU,O-(7-azabenzotraiazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU),2-(6-Chloro-1H-benzotraiazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (HCTU), and the like.

A preferred method of attachment to the Fmoc-PAL-PEG-PS resin may beaccomplished by deprotection of the resin linker with 20% piperidine inDMF, followed by reaction of the N-α-Fmoc protected amino acid, about a5 fold molar excess of the N-α-Fmoc-amino acid, using HBTU: DIEA (1:2)in DMF in a microwave-assisted peptide synthesizer with a 5 min, 75° maxcoupling cycle.

For this Fmoc-based protocol in the microwave-assisted peptidesynthesizer, the N-α-Fmoc amino acid protecting groups are removed with20% piperadine in DMF containing 0.1M 1-hydroxybenzotriazole (HOBt), ina double deprotection protocol for 30 sec and then for 3 min with atemperature maximum set at 75° C. HOBt is added to the deprotectionsolution to reduce aspartimide formation. Coupling of the next aminoacid then employs a five-fold molar excess using HBTU:DIEA (1:2) with a5 min, 75° max. double-coupling cycle.

At the end of the solid phase synthesis the fully protected peptide isremoved from the resin. When the linkage to the resin support is of thebenzyl ester type, cleavage may be effected by means of aminolysis withan alkylamine or fluoroalkylamine for peptides with an alkylamideC-terminus, or by ammonolysis with, for example, ammonia/methanol orammonia/ethanol for peptides with an unsubstituted amide C-terminus, ata temperature between about −10° and 50° C., preferably about 25° C.,for between about 12 and 24 hours, preferably about 18 hours. Peptideswith a hydroxy C-terminus may be cleaved by HF or other strongly acidicdeprotection regimen or by saponification. Alternatively, the peptidemay be removed from the resin by transesterification, e.g., withmethanol, followed by aminolysis or saponification. The protectedpeptide may be purified by silica gel or reverse-phase HPLC.

The side chain protecting groups may be removed from the peptide bytreating the aminolysis product with, for example, anhydrous liquidhydrogen fluoride in the presence of anisole or other carbonium ionscavenger, treatment with hydrogen fluoride/pyridine complex, treatmentwith tris(trifluoroacetyl)boron and trifluoroacetic acid, by reductionwith hydrogen and palladium on carbon or polyvinylpyrrolidone, or byreduction with sodium in liquid ammonia, preferably with liquid hydrogenfluoride and anisole at a temperature between about −10° and +10° C.,preferably at about 0° C., for between about 15 minutes and 2 hours,preferably about 1.5 hours.

For peptides on the benzhydrylamine type resins, the resin cleavage anddeprotection steps may be combined in a single step utilizing liquidhydrogen fluoride and anisole as described above or preferably throughthe use of milder cleavage cocktails. For example, for the PAL-PEG-PSresin, a preferred method is through the use of a double deprotectionprotocol in the microwave-assisted peptide synthesizer using one of themild cleavage cocktails known in the art, such asTFA/water/tri-iso-propylsilane/3,6-dioxa-1,8-octanedithiol (DODT)(92.5/2.5/2.5/2.5) for 18 min at 38° C. each time. Cleavage of alkylglycoside containing materials have shown survival of the alkylglycoside linkage using protocols with TFA/water ratios in the 9/1 to19/1 range. A typical cocktail is 94% TFA: 2% EDT; 2% H₂O; 2% TIS.Typically the fully deprotected product is precipitated and washed withcold (−70° to 4° C.) diethylether, dissolved in deionized water andlyophilized.

The peptide solution may be desalted (e.g. with BioRad AG-3® anionexchange resin) and the peptide purified by a sequence ofchromatographic steps employing any or all of the following types: ionexchange on a weakly basic resin in the acetate form; hydrophobicadsorption chromatography on underivatizedco-poly(styrene-divinylbenzene), e.g. Amberlite® XAD; silica geladsorption chromatography; ion exchange chromatography oncarboxymethylcellulose; partition chromatography, e.g. on Sephadex®G-25; counter-current distribution; supercritical fluid chromatography;or HPLC, especially reversed-phase HPLC on octyl- oroctadecylsilylsilica (ODS) bonded phase column packing.

Also provided herein are processes for preparing covalently modifiedpeptides and/or proteins described herein and pharmaceuticallyacceptable salts thereof, which processes comprise sequentiallycondensing protected amino acids on a suitable resin support, removingthe protecting groups and resin support, and purifying the product, toafford analogs of the physiologically active truncated homologs andanalogs of the covalently modified peptides and/or proteins describedherein. In some embdiments, covalently modified peptides and/or proteinsdescribed herein incorporate alkyl glycoside modifications as definedabove.

Another aspect relates to processes for preparing covalently modifiedpeptides and/or proteins described herein and pharmaceuticallyacceptable salts thereof, which processes comprise the use ofmicrowave-assisted solid phase synthesis-based processes or standardpeptide synthesis protocols to sequentially condense protected aminoacids on a suitable resin support, removing the protecting groups andresin support, and purifying the product, to afford analogs of thephysiologically active peptides, as defined above.

Example 3: N-Terminal Endomorphin-1 Analog—AcLys(1-octylβ-D-glucuronide-6-yl)endomorphin 1 (Ac-Lys(1-octylβ-D-glucuronide-6-yl)-Tyr-Pro-Trp-Phe-NH₂ (AcLys(1-octylβ-D-glucuronide-6-yl)endomorphin 1)

As described above, Fmoc-Tyr(t-Bu)-Pro-Trp(Boc)-Phe-NH-Rink amide MBHAresin is prepared as described in (Koda, Y., et al. (2008) Bioorg MedChem 16: 6286-6296). The resin is Nα-deprotected with piperadine/DMFsolution, washed with solvent and coupled with 2 equivalents ofN-α-Ac,N-ε-(1-octyl β-D-glucuronide-6-yl)-L-lysine using a standardcoupling mixture (e.g. HBTU/DIPEA). Following completion of thecoupling, the peptide is deprotected and cleaved from the resin using adeprotection mixture (95% TFA/5% H₂O or DODT). The solvent is removed bya stream of nitrogen and the crude peptide is precipitated with coldEt₂O, collected, dissolved in 20% acetonitrile and lyophilized.Purification is by reversed phase hplc in a mobile phase containing agradient from H₂O to H₂O/acetonitrile using a 0.1% TFA or NH₄OAc buffersystem. The mixture is subjected to multiple lyophilizations from H₂O.

Example 4: N-Terminal Endomorphin-1 Analog—AcLys(1-octylβ-D-glucuronide-6-yl)endomorphin 1 (Ac-Lys(1-octylβ-D-glucuronide-6-yl)-Tyr-Pro-Trp-Phe-NH₂ (AcLys(1-octylβ-D-glucuronide-6-yl)endomorphin 1)

As described above, Ac-Lys(Boc)-Tyr(t-Bu)-Pro-Trp(Boc)-Phe-NH-Rink amideMBHA resin is prepared as described in. Following completion of thesynthesis, the peptide is deprotected and cleaved from the resin using adeprotection mixture (95% TFA/5% H₂O or DODT). The solvent is removed bya stream of nitrogen and the crude peptide is precipitated with coldEt₂O, collected, dissolved in 20% acetonitrile and lyophilized. Thepeptide, containing a deprotected Lys ε-amino function is coupled with 2equivalents of 1-octyl β-D-glucuronic acid using a standard couplingmixture (e.g. HBTU/DIPEA) in DMF or similar anhydrous aprotic solvent.The solvent is removed in vacuo and the product is lyophilized from 20%acetonitrile or H₂O. Purification is by reversed phase hplc in a mobilephase containing a gradient from H₂O to H₂O/acetonitrile using a 0.1%TFA or NH₄OAc buffer system. The mixture is subjected to multiplelyophilizations from H₂O.

Example 5:2′,6′-dimethyl-L-tyrosyl-prolyl-2′,4′,6′-trimethyl-L-phenylalanyl-Nε-(1′-octylβ-D-glucuronyl)-L-lysine amide (EU-A102)

A 0.3 mmol sample of Fmoc-Rink-Amide resin (0.5 mmol/g) was coupled withthe following sequence of amino acids using a standard DIC/HOBt solidphase coupling protocol (3 equivalent of DIC/HOBt and amino acid):Fmoc-L-lysine(Nε-Alloc); Fmoc-2′,4′,6′-trimethyl-L-phenylalanine;Fmoc-L-proline; Fmoc-2′,4′-dimethyl-L-tyrosine.

The sample of2′,6′-dimethyl-L-tyrosyl-prolyl-2′,4′,6′-trimethyl-L-phenylalanyl-Nε-(Alloc)-L-lysineamide resin was deprotected on the Lys-NF position by incubation withPd(PPh₃)₄ (0.5 eq) and DMBA (20 eq) in DMF/CH₂Cl₂ (1:1) overnight in thedark at room temperature. Following washing by DMF/CH₂Cl₂, the Lys sidechain was acylated with 1′-octyl β-D-glucuronic acid (Carbosynth) inDMF/CH₂Cl₂ through the use of DIC/HOBt. Completion of the coupling waschecked by ninhydrin and the product was washed extensively with CH₂Cl₂.

The product resin (0.77 g) was submitted to final deprotection andcleavage from the resin by treatment with the cleavage cocktail (94%TFA: 2% EDT; 2% H₂O; 2% TIS) for a period of 240 min at roomtemperature. The mixture was treated with Et₂O, to precipitate theproduct and washed extensively with Et₂O to yield 290 mg of crude titlepeptide product after drying in vacuo.

Purification was carried out in two batches by reversed-phase (C18)hplc. The crude peptide was loaded on a 4.1×25 cm hplc column at a flowrate of 15 mL/min (15% organic modifier; acetic acid buffer) and elutedwith a gradient from 15-45% buffer B in 60 min at 50° C. The productfraction was lyophilized to yield 100 mg of the title product peptidewith a purity >96% by analytical hplc/mass spectrometry (M+1peak=911.87). The overall synthesis yield was calculated at 18%.

In a similar manner, but using the reagents 1′-methyl β-D-glucuronicacid and 1′-dodecyl 3-D-glucuronic acid, were prepared the correspondingNε-(1′-methyl β-D-glucuronyl)-L-lysine⁴ (EU-A101) and Nε-(1′-dodecylβ-D-glucuronyl)-L-lysine⁴ (EUA-103) analogs of the title compound.

Analysis was done by HPLC/mass spectrometery in positive ion mode usingthe eluent gradients given in the table below.

Molecular Compound Position Wt Molecular HPLC (min; Name 4 Nε expectedWt found elution) EU-A101 Me 812.95 812.80 13.6 [a] EU-A102 n-octyl911.16 910.87 12.9 [b] EU-A103 n-dodecyl 967.27 966.53 12.5 [c] HPLCgradients in 0.1% TFA [a] 20 to 50% CH₃CN over 30 min. [b] 35 to 65%CH₃CN over 30 min. [c] 40 to 75% CH₃CN over 20 min. HPLC on PhenomenexLuna C18 5 micron 250 × 4.6 mm

Example 6:2′,6′-dimethyl-L-tyrosyl-prolyl-2′,4′,6′-trimethyl-L-phenylalanyl-L-phenylalanyl-Nε-(1′-dodecylβ-D-glucuronyl)-L-lysine amide (EU-A106)

In a similar manner to that given for the solid phase synthesis andlysine side chain modification in example 5, given above, but using1-dodecyl β-D-glucuronic acid (Milkereit, G., et al. (2004) Chem PhysLipids 127: 47-63) for lysine Nε-acylation, was prepared the titlepeptide as a crude product. Following reversed phase hplc purificationas above one obtains the title product as a white powder, of 96.1%purity by hplc/mass spectrometry (M+1=1114.8).

In a similar manner, but using the reagent 1′-methyl β-D-glucuronicacid, was prepared the corresponding Nε-(1′-methylβ-D-glucuronyl)-L-lysine⁵ (EUA-105) analog of the title compound.

Analysis was done by HPLC/mass spectrometery in positive ion mode usingthe gradient eluents given in the table below.

Molecular Compound Position Wt Molecular Wt HPLC Name 5 Nε expectedfound (min; elution) EU-A105 Me 960.12 959.60  8.8 [d] EU-A106 n-dodecyl1114.44 1113.80 11.6 [e] HPLC gradients in 0.1% TFA [d] 30 to 60% CH₃CNover 20 min. [e] 45 to 75% CH₃CN over 20 min. HPLC on Phenomenex LunaC18 5 micron 250 × 4.6 mm

Example 7: 2′,6′-dimethyl-L-tyrosyl-Nε-(1′-dodecylβ-D-glucuronyl)-D-lysyl-2′,4′,6′-trimethyl-L-phenylalanyl-L-phenylalanine-amide(EU-A108)

In a similar manner as that given in Example 6 was prepared the titlepeptide as a white powder of 95.8% purity by hplc/mass spectrometry(M=1017.07).

In a similar manner, but using the reagent 1′-methyl (β-D-glucuronicacid, was prepared the corresponding Nε-(1′-methylβ-D-glucuronyl)-L-lysine (EU-A107) analog of the title compound.

Analysis was done by HPLC/mass spectrometry in positive ion mode usingthe eluent gradients given in the table below.

Molecular Compound Position Wt Molecular Wt HPLC Name 2 Nε expectedfound (min; elution) EU-A107 Me 862.01 862.73 11.6 [f] EU-A108 n-dodecyl1017.32 1017.07 11.9 [g] HPLC gradients in 0.1% TFA [f] 25 to 55% CH₃CNover 20 min. [g] 50 to 80% CH₃CN over 20 min. HPLC on Phenomenex LunaC18 5 micron 250 × 4.6 mm

Example 8:2′,6′-dimethyl-L-tyrosyl-L-1,2,3,4-tetrahydroisoquinoline-3-carbonyl-L-phenylalanyl-Nε-(1′-methylβ-D-glucuronyl)-L-lysyl-amide (EU-A178)

In a similar manner as that given in Example 6 was prepared the titlepeptide as a white powder of 95.5% purity by hplc/mass spectrometry(M=832.33).

In a similar manner, but using the reagent 1′-dodecyl β-D-glucuronicacid, was prepared the corresponding Nε-(1′-dodecylβ-D-glucuronyl)-L-lysine analog (EU-A179) of the title compound. In asimilar manner, but using the corresponding 1-alkyl glucuronic acidreagents are made the corresponding peptides of the invention, EU-A180,EU-A181, EU-A182, EU-A183, EU-A184, EU-A185, and the like.

Analysis was done by HPLC/mass spectrometry in positive ion mode usingthe eluent gradients given in the table below.

Molecular Compound Position Wt Molecular Wt HPLC Name 4 Nε expectedfound (min; elution) EU-A178 Me 832.91 832.33 11.2 [h] EU-A179 n-dodecyl987.23 986.47 10.7 [f] EU-A180 n-octyl 931.12 930.67 10.1 [i] HPLCgradients in 0.1% TFA [f] 25 to 55% CH₃CN over 20 min. [h] 20 to 50%CH₃CN over 20 min. [i] 35 to 65% CH₃CN over 20 min HPLC on PhenomenexLuna C18 5 micron 250 × 4.6 mm

Example 9:2′,6′-dimethyl-L-tyrosyl-L-1,2,3,4-tetrahydroisoquinoline-3-carbonyl-L-phenylalanyl-L-phenylalanyl-Nε-(1′-methylβ-D-glucuronyl)-L-lysyl-amide (EU-A189)

In a similar manner as that given in Example 6 was prepared the titlepeptide as a white powder of 98.99% purity by hplc/mass spectrometry(M=979.53).

In a similar manner, but using the reagent 1′-dodecyl β-D-glucuronicacid, was prepared the corresponding Nε-(1′-dodecylβ-D-glucuronyl)-L-lysine (EU-A190) in position 5 analog of the titlecompound.

Analysis was done by HPLC/mass spectrometry in positive ion mode usingthe eluent gradients given in the table below.

Molecular Compound Position Wt Molecular Wt HPLC Name 5 Nε expectedfound (min; elution) EU-A189 Me 980.09 979.53 10.9 [e] EU-A190 n-dodecyl1134.41 1134.53 12.5 [e] HPLC gradients in 0.1% TFA [e] 45 to 75% CH₃CNover 20 min. HPLC on Phenomenex Luna C18 5 micron 250 × 4.6 mm

Example 10: Additional Analogs of the Invention

In a similar manner as that given in Example 6, but using thecorresponding 1-alkyl β-D-glucuronic acid reagent are prepared theadditional peptides of the invention as white powders of greater than95% purity by hplc/mass spectrometry.

Analysis is done by HPLC/mass spectrometry in positive ion mode usingthe appropriate eluent gradients such as those given in the table below.

Molecular Compound Position Wt Molecular HPLC Name 3 or 4 Nε expected Wtfound (min; elution) EU-A600 Me 918.02 918.00 10.7 [h] EU-A601 n-octyl1016.23 1015.87 10.1 [i] EU-A615 Me 832.91 832.67 11.0 [h] EU-A620 Me923.06 922.73  9.5 [d] EU-A639 Me 923.06 922.80 11.5 [f] HPLC gradientsin 0.1% TFA [d] 30 to 60% CH₃CN over 20 min. [e] 45 to 75% CH₃CN over 20min. [h] 20 to 50% CH₃CN over 20 min. [f] 25 to 55% CH₃CN over 20 min.[i] 35 to 65% CH₃CN over 20 min HPLC on Phenomenex Luna C18 5 micron 250× 4.6 mm

Example 11. General Oxidation Method for Uronic Acids

To a solution of 1-dodecyl J-D-glucopyranoside (Carbosynth) [2.0 g, 5.74mmol] in 20 mL of acetonitrile and 20 mL of DI water was added(diacetoxyiodo)benzene (Fluka) [4.4 g, 13.7 mmol] and TEMPO(SigmaAldrich) [0.180 g, 1.15 mmol]. The resulting mixture was stirredat room temperature for 20 h. The reaction was followed by massspectrometry (for example LCQ ESI) and upon completion, the reactionmixture was diluted with water and lyophilized to dryness to give 1.52 g(crude yield 73.1%) of the crude product, 1-dodecyl β-D-glucuronic acid,as a white powder, which was used directly for the solid phase synthesiswithout further purification. In a like manner, but using thecorresponding 1-tetradecyl, 1-hexadecyl, and 1-octadecylβ-D-glucopyranosides (purchased from Anatrace, Maumee, Ohio) wereprepared the desired alkyl saccharide uronic acids used to make theproducts and reagents described herein. This product was previouslyprepared by an alternative process using NaOCl as oxidant and also hasbeen used for longer alkyl groups. For longer alkyl groups 1,4-dioxanewas used instead of acetonitrile and the temperature was raised as highas 30° C.

In a like manner, but using, for example, the corresponding 1-octyl,1-decyl, 1-undecyl, 1-tetradecyl, 1-hexadecyl, and 1-octadecylglycosides (purchased from Anatrace, Maumee, Ohio) were prepared thedesired 1-alkyl saccharide uronic acids which were used to make theproducts and reagents described herein. In a like manner, but using, forexample, the corresponding 1-octyl, 1-decyl, 1-undecyl, 1-tetradecyl,1-hexadecyl, and 1-octadecyl β-D-melibiosides or β-D-maltosides(purchased from Anatrace, Maumee, Ohio) were prepared the desired1-alkyl disaccharide uronic acids which were used to make the productsand reagents described herein. In each case the corresponding alphaglycosides can be treated similarly.

Example 12: Cellular Assay of the Compounds

Compounds were weighed precisely in an amount of approximately 2 mg andassayed in standard cellular assays by the contract researchorganization Cerep, Inc. (Pullman, Wash.) as executed at theirsubsidiary, Cerep SA (Le Bois l'Eveque, France). The readout is theamount of cAMP generated in the cells treated with the test compounds,in either agonist or antagonist mode. The assays used were the mu opioidreceptor cellular assay (MOP in agonist and antagonist mode), the delta2opioid receptor cellular assay (DOP in agonist and antagonist mode) andthe kappa opioid receptor cellular assay (KOP in agonist and antagonistmode). The assays used are described in Wang, J. B., et al. (1994) FEBSLett 338: 217-222, Law, P. Y. and Loh, H. H. (1993) Mol Pharmacol 43:684-693, and Avidor-Reiss, T., et al. (1995) FEBS Lett 361: 70-74. Thestimulant for the cells in the DOR antagonist assay was 3×10E-8M DPDPE,a well-accepted DOR literature standard.

For the series of compounds EU-A101 to EU-A103, where the hydrophobicportion of the surfactant (1-alkyl glucuronic acid) varies in lengthfrom C1 to C12, the character of the receptor selectivity and activation(see below) varies from full agonist (C1) to pure antagonist (C12). Noactivity was seen in cells used for DOP or KOP assays, thus showingselectivity for the mu opioid receptor as agonists. This behaviordemonstrates the ability of the modifications described herein to varythe fundamental properties of the receptor interactions. Modificationselsewhere in the molecule (e.g., using amino acid analogs) are used tofurther modify the potency and character of the interaction of the drugcandidates.

MOP MOP Compound agonist antagonist Name Structure EC₅₀ (nM) IC₅₀ (nM)Characterization EU-A101 Me 13 nc pure agonist EU-A102 n-octyl 100  100partial agonist EU-A103 n-dodecyl Nc  86 pure antagonist EU-A107 Me 60nc Agonist

DOP antagonistic activity was assessed by inhibition of the stimulatorycAMP response of 3×10E-8M DPDPE.

MOP Compound agonist DOP antagonist Name Structure EC₅₀ (nM) IC₅₀ (M)Characterization EU-A178 Me  15 <10E−10 Pure MOP Ag; pure DOP antagEU-A179 n-dodecyl § NT Pure MOP Ag; pure DOP antag EU-A180 n-octyl  36<10E−10 Pure MOP Ag; pure DOP antag EU-A189 Me  41 NT Pure MOP Ag; pureDOP antag EU-A190 n-dodecyl § NT Pure MOP Ag; pure DOP antag EU-A600 Me110 <10E−10 Pure MOP Ag; pure DOP antag EU-A601 n-octyl § <10E−10 PureMOP Ag; pure DOP antag EU-A615 Me 410  3.1E−10 Pure MOP Ag; pure DOPantag EU-A620 Me 480 <10E−10 Pure MOP Ag; pure DOP antag EU-A639 Me 280<10E−10 Pure MOP Ag; pure DOP antag § indicates solubility issues duringdissolution, NT means not tested, nc means not calculable

Example 13: In Vivo Assay of the Compounds

λ-Carrageenan-Induced Inflammatory Pain Model:

Adult male Sprague-Dawley rats were briefly anesthetized withisoflurane, and unilateral hind paw inflammatory pain was induced byinjecting 3.5 g/100 μl of λ-carrageenan. A λ-carrageenan injection intothe left hind paw produces redness and edema. These alterations wereobserved within 6 h after induction of inflammation and peaked by 24 h.Further, the changes were confined to the carrageenan treated paw.Paired t test showed a significant increase in paw volume (p<0.0001) anddecrease in paw pressure threshold (p<0.001) following carrageenantreatment.

Changes in Paw Pressure Thresholds (PPT), a measurement of nociceptionand antinociception, was evaluated using a digital Randall-Selittomethod. Briefly, animals are gently restrained and incremental pressure(maximum 350 g) was applied onto the dorsal surface of the hindpaw. Thepressure required to elicit paw withdrawal, PPT, was determined.Paw-thickness, the indicator of inflammation, was measured using digitalcalipers. 24 h after induction of inflammation, rats received eithersaline, positive controls (DAMGO (8-32 μg) or fentanyl (0.4-0.8 μg)) ortest peptides in 100 μl volume i.pl. The antinociception was measured atdifferent time points (5 to 120 min or 5 hr to 24 hr) after drugadministration. Baseline measures were taken before administering drugto confirm the development of inflammation and nociception. Eu-A178, forexample, showed statistically significant increases in PPT beyond 30min.

Example 14: Uses of the Compounds

The covalently modified peptides and/or proteins described herein areuseful for the prevention and treatment of a variety of diseasesdepending on which class is being considered. For example, covalentlymodified peptides and/or proteins described herein are indicated for theprophylaxis and therapeutic treatment of chronic and acute pain andother MOR- or DOR-related disease states. Applications for sunburn,pruritus, cancer, immune function, inflammation, cardiovascular diseasealso have been documented (Lazarus, L. H., et al. (2012) Expert OpinTher Patents 22: 1-14).

Representative delivery regimens include oral, parenteral (includingsubcutaneous, intramuscular and intravenous injection), rectal, buccal(including sublingual), transdermal, inhalation ocular and intranasal.An attractive and widely used method for delivery of peptides entailssubcutaneous injection of a controlled release injectable formulation.Other administration routes for the application of the covalentlymodified peptides and/or proteins described herein are oral,subcutaneous, intranasal and inhalation administration.

Example 15. Pharmaceutical Usage for Treatment of Pain

A human patient with acute or chronic pain is treated with EU-A178 byintranasal administration (200 μL) from a standard atomizer used in theart of a solution of the pharmaceutical agent in physiological salinecontaining from 0.5 to 10 mg/mL of the pharmaceutical agent andcontaining standard excipients such as benzyl alcohol. The treatment isrepeated as necessary for the alleviation of pain. Alternatively asolution of EU-A178, and selected excipients, in an evaporating solventcontaining such as a hydrofluoroalkane is administered intranasally byMDI as needed to alleviate pain. Alternatively an aqueous solution ofEU-A178, with selected excipients, is administered by subcutaneousinjection as needed to alleviate pain.

The effect of treatment is determined from evaluation of patients,including quality of life questionaires. Pain scales are based onself-report, observational (behavioral), or physiological data. Somepain scales suitable for use in clinical setting include Alder HeyTriage Pain Score, Brief Pain Inventory (BPI), Dallas PainQuestionnaire, Dolorimeter Pain Index (DPI), McGill Pain Questionnaire(MPQ), Numerical 11 point box (BS-11), Numeric Rating Scale (NRS-11),Roland-Morris Back Pain Questionnaire, Visual analog scale (VAS) or thelike.

In a similar manner, administration of an adjusted amount bytransbuccal, intravaginal, inhalation, subcutaneous, intravenous,intraocular, or oral routes is tested to determine relief from pain.

Example 2-1: Reagents—N-α-Fmoc, N-ε-(1-octylβ-D-glucuronide-6-yl)-L-lysine

In an oven-dried 250 mL Erlenmeyer flask is placed 1-octylβ-D-glucuronic acid (Carbosynth Ltd., 3.06 g, 10 mmol), 50 mL anhydrousDMF, and anhydrous 1-hydroxybenzotriazole (1.62 g, 12 mmol). A chilled(4° C.) solution of N, N′-dicyclohexylcarbodiimide (2.48 g, 12 mmol) in50 mL of DMF is added, with stirring, and the reaction is allowed toproceed for 5 min. The copious white precipitate of N,N′-dicyclohexylurea is filtered on a fritted glass funnel and thefiltrate is added to a solution of N-α-Fmoc-L-lysine (3.68 g, 10 mmol)in 25 ml anhydrous DMF. The reaction is allowed to proceed for 25 minwith warming to room temp or until the ninhydrin color is very faint.The reaction mixture is filtered, stripped to dryness and crystallizedfrom MeOH/Et₂O by dissolution in MeOH and slow dilution to the cloudpoint with Et₂O, followed by refrigeration. Further purification can beachieved by silica gel chromatography using a solvent gradient fromEtOAc to EtOAc/EtOH/AcOH.

In a similar manner, but substituting N-α-Boc-L-lysine is obtainedN-α-Boc,N-ε-(1-octyl β-D-glucuronide-6-yl)-L-lysine, suitable forN-terminal incorporation and cleavage to a free N-Terminus. In a similarmanner, but substituting N-α-Ac-L-lysine is obtained N-α-Ac,N-ε-(1-octyl β-D-glucuronide-6-yl)-L-lysine, suitable for incorporationat the N-terminus of a peptide with a blocked N-terminus. In a similarmanner, but substituting the appropriate amount of N-α-Fmoc-L-ornithineis obtained N-α-Fmoc,N-δ-(1-octyl β-D-glucuronide-6-yl)-L-ornithine. Ina similar manner but substituting other N-mono-protected diamino acidsone obtains the corresponding reagents. Alternatively, use of atransient Me₃Si ester protecting group during the coupling and withoutpreactivation of the 1-octyl β-D-glucuronic acid provides a facile routeto the formation of the reagents. The transient Me₃Si ester is producedby reaction of the Fmoc-Lys-OH with an equimolar amount ofN,O-bis(trimethylsilyl)acetamide in dichloromethane (CH₂Cl₂). Theorganic layer contains the desired reagent as a solution in CH₂Cl₂ readyfor coupling with the 1-alkyl glucoronide as above. The filteredreaction mixture is washed with aqueous NaHSO₄ to hydrolyze the Me₃Siester, dried over MgSO₄ and solvent is removed.

Similarly, but using peracetyl or perbenzoyl 1-octyl β-D-glucuronic acidone obtains the Ac, or Bz protected form of the reagents (e.g.2,3,4-trisacetyl 1-octyl β-D-glucuronic acid, and the like, formed bytreatment with Ac₂O). Such reagents have increased stability during acidcleavage from the resin and are used when instability duringdeprotection is detected, see (Kihlberg, J., et al. (1997) MethodsEnzymol 289: 221-245) and references therein. Final deprotection of suchproducts is carried out by base-catalyzed transesterification aftercleavage, by use of MeOH/NH₃, MeOH/NaOMe, MeOH/NH₂NH₂, as describedabove.

Example 2-2: Synthetic Peptide Analogs

In general, peptide synthesis methods involve the sequential addition ofprotected amino acids to a growing peptide chain. Normally, either theamino or carboxyl group of the first amino acid and any reactive sidechain group are protected. This protected amino acid is then eitherattached to an inert solid support, or utilized in solution, and thenext amino acid in the sequence, also suitably protected, is added underconditions amenable to formation of the amide linkage. After all thedesired amino acids have been linked in the proper sequence, protectinggroups and any solid support are removed to afford the crude peptide.The peptide is desalted and purified chromatographically.

A preferred method of preparing the analogs of the physiologicallyactive truncated peptides, having fewer than about fifty amino acids,involves solid phase peptide synthesis. In this method the α-amino (Nα)functions and any reactive side chains are protected by acid- orbase-sensitive groups. The protecting group should be stable to theconditions of peptide linkage formation, while being readily removablewithout affecting the extant peptide chain. Suitable a-amino protectinggroups include, but are not limited to t-butyloxycarbonyl (Boc),benzyloxycarbonyl (Cbz), o-chlorobenzyloxycarbonyl,biphenylisopropyloxycarbonyl, t-amyloxycarbonyl (Amoc),isobornyloxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxy-carbonyl,o-nitrophenylsulfenyl, 2-cyano-t-butoxycarbonyl,9-fluorenyl-methoxycarbonyl (Fmoc) and the like, preferably Boc or morepreferably, Fmoc. Suitable side chain protecting groups include, but arenot limited to: acetyl, benzyl (Bzl), benzyloxymethyl (Bom), Boc,t-butyl, o-bromobenzyloxycarbonyl, t-butyl, t-butyldimethylsilyl,2-chlorobenzyl (Cl-z), 2,6-dichlorobenzyl, cyclohexyl, cyclopentyl,isopropyl, pivalyl, tetrahydropyran-2-yl, tosyl (Tos),2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), trimethylsilyland trityl. A preferred Nα-protecting group for synthesis of thecompounds is the Fmoc group. Preferred side chain protecting groups areO-t-Butyl group for Glu, Tyr, Thr, Asp and Ser; Boc group for Lys andTrp side chains; Pbf group for Arg; Trt group for Asn, Gln, and His. Forselective modification of a Lys residue, orthogonal protection with aprotecting group not removed by reagents that cleave the Fmoc or t-butylbased protecting groups is preferred. Preferred examples formodification of the Lys side chain include, but are not limited to,those removed by hydrazine but not piperidine; for example1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde) or1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) andallyloxycarbonyl (Alloc).

The Fmoc-Lys(ivDde) or Fmoc-Lys(Dde) protecting group scheme ispreferred in cases where side chain lactam formation is desired(Houston, M. E., Jr., et al. (1995) J Pept Sci 1: 274-282; Murage, E.N., et al. (2010) J Med Chem), since in this case Fmoc-Glu(O-Allyl) andFmoc-Lys(Alloc) can be incorporated and used to provide transientprotection, then deprotected for lactam formation while the Lys(Dde)protecting group remains for later removal and reaction with thefunctionalized surfactant. The side chain lactam between acidic andbasic residue (e.g. Glu and Lys) is carried out after removal of theallyl-based protection by activation of the carboxyl side chain functionwith N,N′-diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) or2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate(HBTU)/N,N-di-isopropylethylamine (DIEA), using standard protocols wellknown to the art.

In solid phase synthesis, the C-terminal amino acid is first attached toa suitable resin support. Suitable resin supports are those materialswhich are inert to the reagents and reaction conditions of the stepwisecondensation and deprotection reactions, as well as being insoluble inthe media used. Examples of commercially available resins includestyrene/divinylbenzene resins modified with a reactive group, e.g.,chloromethylated co-poly-(styrene-divinylbenzene), hydroxymethylatedco-poly-(styrene-divinylbenzene), and the like. Benzylated,hydroxymethylated phenylacetamidomethyl (PAM) resin is preferred for thepreparation of peptide acids. When the C-terminus of the compound is anamide, a preferred resin isp-methylbenzhydrylamino-co-poly(styrene-divinyl-benzene) resin, a 2,4dimethoxybenzhydrylamino-based resin (“Rink amide”),4-Hydroxymethylphenoxyacetyl aminomethyl resin (HMP Am) and the like. Anespecially preferred support for the synthesis of larger peptides arecommercially available resins containing PEG sequences grafted ontoother polymeric matricies, such as the Rink Amide-PEG and PAL-PEG-PSresins (Applied Biosystems) or similar resins designed for peptide amidesynthesis using the Fmoc protocol. Thus in certain cases it is desirableto have an amide linkage to a PEG chain. It those cases it is convenientto link an N-Fmoc-amino-PEG-carboxylic acid to the amide forming resinabove (e.g. Rink amide resin and the like). The first amino acid of thechain can be coupled as an N-Fmoc-amino acid to the amino function ofthe PEG chain. Final deprotection will yield the desiredPeptide-NH-PEG-CO—NH₂ product.

Attachment to the PAM resin may be accomplished by reaction of the Nαprotected amino acid, for example the Boc-amino acid, as its ammonium,cesium, triethylammonium, 1,5-diazabicyclo-[5.4.0]undec-5-ene,tetramethylammonium, or similar salt in ethanol, acetonitrile,N,N-dimethylformamide (DMF), and the like, preferably the cesium salt inDMF, with the resin at an elevated temperature, for example betweenabout 40° and 60° C., preferably about 50° C., for from about 12 to 72hours, preferably about 48 hours. This will eventually yield the peptideacid product following acid cleavage or an amide following aminolysis.

The Nα-Boc-amino acid may be attached to the benzhydrylamine or HPM Amresin by means of, for example, an N,N′-diisopropylcarbodiimide(DIC)/1-hydroxybenzotriazole (HOBt) mediated coupling for from about 2to about 24 hours, preferably about 2 hours at a temperature of betweenabout 10° and 50° C., preferably 25° C. in a solvent such as CH₂Cl₂ orDMF, preferably CH₂Cl₂.

For Boc-based protocols, the successive coupling of protected aminoacids may be carried out by methods well known in the art, typically inan automated peptide synthesizer. Following neutralization withtriethylamine, N,N-di-isopropyl ethylamine (DIEA), N-methylmorpholine(NMM), collidine, or similar base, each protected amino acid isintroduced in approximately about 1.5 to 2.5 fold molar excess and thecoupling carried out in an inert, nonaqueous, polar solvent such asCH₂Cl₂, DMF, N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA), ormixtures thereof, preferably in dichloromethane at ambient temperature.For Fmoc-based protocols no acid is used for deprotection but a base,preferably DIEA or NMM, is usually incorporated into the couplingmixture. Couplings are typically done in DMF, NMP, DMA or mixedsolvents, preferably DMF. Representative coupling agents areN,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropyl-carbodiimide (DIC)or other carbodiimide, either alone or in the presence of HOBt, O-acylureas, benzotriazol-1-yl-oxytris(pyrrolidino)phosphoniumhexafluorophosphate (PyBop), N-hydroxysuccinimide, otherN-hydroxyimides, or oximes. Alternatively, protected amino acid activeesters (e.g. p-nitrophenyl, pentafluorophenyl and the like) orsymmetrical anhydrides may be used. Preferred coupling agents are of theaminium/uronium (alternative nomenclatures used by suppliers) class suchas 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (HBTU),O-(7-azabenzotraiazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU),2-(6-Chloro-1H-benzotraiazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (HCTU), and the like.

A preferred method of attachment to the Fmoc-PAL-PEG-PS resin may beaccomplished by deprotection of the resin linker with 20% piperidine inDMF, followed by reaction of the N-α-Fmoc protected amino acid, about a5 fold molar excess of the N-α-Fmoc-amino acid, using HBTU:di-isopropylethylamine (DIEA) (1:2) in DMF in a microwave-assistedpeptide synthesizer with a 5 min, 75° max coupling cycle.

For this Fmoc-based protocol in the microwave-assisted peptidesynthesizer, the N-α-Fmoc amino acid protecting groups are removed with20% piperidine in DMF containing 0.1M 1-hydroxybenzotriazole (HOBt), ina double deprotection protocol for 30 sec and then for 3 min with atemperature maximum set at 75° C. HOBt is added to the deprotectionsolution to reduce aspartimide formation. Coupling of the next aminoacid then employs a five-fold molar excess using HBTU:DIEA (1:2) with a5 min, 75° max. double-coupling cycle.

At the end of the solid phase synthesis the fully protected peptide isremoved from the resin. When the linkage to the resin support is of thebenzyl ester type, cleavage may be effected by means of aminolysis withan alkylamine or fluoroalkylamine for peptides with an alkylamideC-terminus, or by ammonolysis with, for example, ammonia/methanol orammonia/ethanol for peptides with an unsubstituted amide C-terminus, ata temperature between about −10° and 50° C., preferably about 25° C.,for between about 12 and 24 hours, preferably about 18 hours. Peptideswith a hydroxy C-terminus may be cleaved by HF or other strongly acidicdeprotection regimen or by saponification. Alternatively, the peptidemay be removed from the resin by transesterification, e.g., withmethanol, followed by aminolysis or saponification. The protectedpeptide may be purified by silica gel or reverse-phase HPLC.

The side chain protecting groups may be removed from the peptide bytreating the aminolysis product with, for example, anhydrous liquidhydrogen fluoride in the presence of anisole or other carbonium ionscavenger, treatment with hydrogen fluoride/pyridine complex, treatmentwith tris(trifluoroacetyl)boron and trifluoroacetic acid, by reductionwith hydrogen and palladium on carbon or polyvinylpyrrolidone, or byreduction with sodium in liquid ammonia, preferably with liquid hydrogenfluoride and anisole at a temperature between about −10° and +10° C.,preferably at about 0° C., for between about 15 minutes and 2 hours,preferably about 1.5 hours.

For peptides on the benzhydrylamine type resins, the resin cleavage anddeprotection steps may be combined in a single step utilizing liquidhydrogen fluoride and anisole as described above or preferably throughthe use of milder cleavage cocktails. For example, for the PAL-PEG-PSresin, a preferred method is through the use of a double deprotectionprotocol in the microwave-assisted peptide synthesizer using one of themild cleavage cocktails known in the art, such asTFA/water/tri-iso-propylsilane/3,6-dioxa-1,8-octanedithiol (DODT)(92.5/2.5/2.5/2.5) for 18 min at 38° C. each time. Cleavage of alkylglycoside containing materials have shown survival of the alkylglycoside linkage using protocols with TFA/water ratios in the 9/1 to19/1 range. A typical cocktail is 94% TFA: 2% EDT; 2% H₂O; 2% TIS.Typically the fully deprotected product is precipitated and washed withcold (−70° to 4° C.) Et₂O, dissolved in deionized water and lyophilized.

The peptide solution may be desalted (e.g. with BioRad AG-3® anionexchange resin) and the peptide purified by a sequence ofchromatographic steps employing any or all of the following types: ionexchange on a weakly basic resin in the acetate form; hydrophobicadsorption chromatography on underivatizedco-poly(styrene-divinylbenzene), e.g. Amberlite® XAD; silica geladsorption chromatography; ion exchange chromatography oncarboxymethylcellulose; partition chromatography, e.g. on Sephadex®G-25; counter-current distribution; supercritical fluid chromatography;or HPLC, especially reversed-phase HPLC on octyl- oroctadecylsilylsilica (ODS) bonded phase column packing.

Also provided herein are processes for preparing covalently modifiedpeptides and/or proteins described herein and pharmaceuticallyacceptable salts thereof, which processes comprise sequentiallycondensing protected amino acids on a suitable resin support, removingthe protecting groups and resin support, and purifying the product, toafford analogs of the physiologically active truncated homologs andanalogs of the covalently modified peptides and/or proteins describedherein. In some embodiments, covalently modified peptides and/orproteins described herein incorporate alkyl glycoside modifications asdefined above.

Another aspect relates to processes for preparing covalently modifiedpeptides and/or proteins described herein and pharmaceuticallyacceptable salts thereof, which processes comprise the use ofmicrowave-assisted solid phase synthesis-based processes or standardpeptide synthesis protocols to sequentially condense protected aminoacids on a suitable resin support, removing the protecting groups andresin support, and purifying the product, to afford analogs of thephysiologically active peptides, as defined above.

Example 2-3. General Oxidation Method for Uronic Acids

To a solution of 1-dodecyl β-D-glucopyranoside (Carbosynth) [2.0 g, 5.74mmol] in 20 mL of acetonitrile and 20 mL of DI water was added(diacetoxyiodo)benzene (Fluka) [4.4 g, 13.7 mmol] and TEMPO(SigmaAldrich) [0.180 g, 1.15 mmol]. The resulting mixture was stirredat room temperature for 20 h. The reaction was followed by massspectrometry (for example LCQ ESI) and upon completion, the reactionmixture was diluted with water and lyophilized to dryness to give 1.52 g(crude yield 73.1%) of the crude product, 1-dodecyl β-D-glucuronic acid,as a white powder, which was used directly for the solid phase synthesiswithout further purification. This product was previously prepared by analternative process using NaOCl as oxidant, as described in thespecification, and also has been used for longer alkyl groups. In a likemanner, but using the corresponding 1-tetradecyl, 1-hexadecyl, and1-octadecyl β-D-glucopyranosides (purchased from Anatrace, Maumee, Ohio)were prepared the desired alkyl saccharide uronic acids used to make theproducts and reagents described herein. For longer alkyl groups1,4-dioxane was used instead of acetonitrile and the temperature wasraised as high as 30° C.

In a like manner, but using, for example, the corresponding 1-octyl,1-decyl, 1-undecyl, 1-tetradecyl, 1-hexadecyl, and 1-octadecylglycosides (purchased from Anatrace, Maumee, Ohio) were prepared thedesired 1-alkyl saccharide uronic acids which were used to make theproducts and reagents described herein. In a like manner, but using, forexample, the corresponding 1-octyl, 1-decyl, 1-undecyl, 1-tetradecyl,1-hexadecyl, and 1-octadecyl β-D-melibiosides or β-D-maltosides(purchased from Anatrace, Maumee, Ohio) were prepared the desired1-alkyl disaccharide uronic acids which were used to make the productsand reagents described herein. In each case the corresponding alphaglycosides can be treated similarly.

Example 2-4: Preparation of PTHrP Analog EU-204

A sample ofFmoc-Ac5c-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Arg-Ala-Arg-Trp-Ile-Gln-Lys(Alloc)-Rinkamide resin was deprotected on the Lys-N-epsilon position by incubationwith Pd(PPh3)4 (0.5 eq) and DMBA (20 eq) in DMF/CH₂Cl₂ (1:1) overnightin the dark at room temperature. Following washing by DMF/CH₂Cl₂, theLys side chain was acylated with 1′-octyl dodecyl β-D-glucuronic acid(Carbosynth) in DMF/CH₂Cl₂ through the use of DIC/HOBt. Completion ofthe coupling was checked by ninhydrin and the product was washedextensively with CH₂Cl₂.

The product resin was submitted to final deprotection and cleavage fromthe resin by treatment with the cleavage cocktail (94% TFA: 2% EDT; 2%H₂O; 2% TIS) for a period of 240 min at room temperature. The mixturewas treated with Et₂O, to precipitate the product and washed extensivelywith Et₂O to yield the crude title peptide product after drying invacuo.

Purification was carried out in two batches by reversed phase (C18)hplc. The crude peptide was loaded on a 4.1×25 cm hplc column at a flowrate of 15 mL/min (15% organic modifier; acetic acid buffer) and elutedwith a gradient from 15-45% buffer B in 60 min at 50° C. The productfraction was lyophilized to yield the title product peptide with apurity >97% by analytical hplc (12.0 min; 35-65% CH₃CN in 0.1% TFA)/massspectrometry (M+1 peak=2473.9).

The corresponding 1-methyl, 1-octyl, 1-decyl, 1-dodecyl, 1-tetradecyl,1-hexadecyl, 1-octadecyl and 1-eicosyl analogs are prepared using thecorresponding glucouronic acids, prepared as described above.Alternatively, the 1-alkyl glucuronyl, or other uronic acylated analogs,may be prepared by initial purification of the deprotected or partiallydeprotected peptide followed by acylation by the desired uronic acidreagent.

Analysis was done by HPLC/mass spectrometry in positive ion mode usingthe eluent gradients given in the table below.

Molecular Compound Wt Molecular HPLC Name Expected Wt Found (min;elution) EU-201 2278.63 2278.14 14.1 [a] EU-202 2376.86 2376.80 11.2 [b]EU-203 2432.97 2432.40 14.1 [c] EU-204 2472.00 2471.86 12.0 [d] EU-2052343.87 2344.26  8.0 [e] EU-207 2557.12 2557.06 13.2 [c] EU-232 2642.232642.14 12.6 [c] EU-251 2941.56 2942.26 13.1 [c] EU-260 2967.61 2966.6613.9 [c] EU-283 2881.47 2882.26 11.0 [c] EU-284 3640.39 3640.00 11.9 [c]EU-286 2670.47 2669.86  6.3 [e] EU-287 2698.47 2697.74  8.4 [e] EU-2882726.47 2726.26 10.9 [f] HPLC gradients in 0.1% TFA [a] 20 to 50% CH₃CNover 30 min. [b] 25 to 55% CH₃CN over 20 min. [c] 30 to 60% CH₃CN over20 min. [d] 35 to 65% CH₃CN over 20 min. [e] 40 to 70% CH₃CN over 20min. [f] 45 to 75% CH₃CN over 20 min. HPLC on Phenomenex Luna C18 5micron 250 × 4.6 mm.

Example 2-5: Cellular Assay of the Compounds

Compounds were weighed precisely in an amount of approximately 1 mg andassayed in standard cellular assays (cerep SA). The readout is theamount of cAMP generated in the cells treated with the test compounds,in either agonist or antagonist mode. The PTH1 cellular assay used isdescribed in Orloff, J. J., et al. (1992) Endocrinol 131: 1603-1611.

For the series of compounds EU-201 to EU-203, where the hydrophobicportion of the surfactant (1-alkyl glucuronic acid) varies in lengthfrom C1 to C12, the cellular response increases in potency and efficacywith the increased chain length. All of the analogs were agonists.Further substitutions led to molecules with an EC₅₀ similar to PTH1-34,but with super-agonistic activity (e.g. EU-232) and such molecules haveimportant applications in medicine. Additional analogs are designed tohave very prolonged duration of action in vivo (that is EU-286, EU-287,and EU-288). In this assay, PTHrP (coded sample) had an EC50 of 2.9 nMand a maximal response of 100% while the internal standard, PTH had EC50of 1.4 nM and maximal response of 105%. Compounds were dissolved inwater and diluted in assay buffer containing 1% bovine serum albumin.The following table shows potency and efficacy of certain peptideproducts described herein.

Maximal Compound EC50 response Name Structure (nM) (% PTHrP)Characterization EU-201 1-Me 40 115 agonist EU-202 1-octyl 25 118agonist EU-203 1-dodecyl 25 129 agonist EU-204 1-dodecyl 20 135 agonistEU-205 1-dodecyl 40 115 agonist EU-207 1-dodecyl 40 110 agonist EU-2321-dodecyl 2.4 145 agonist EU-251 1-dodecyl 4.3 135 agonist EU-2601-dodecyl 2.2 120 agonist EU-283 1-dodecyl 5.2 100 agonist EU-2841-dodecyl 62 110 agonist EU-287 1-hexadecyl <1 128 agonist EU-2881-octadecyl <1 130 agonist

When tested in antagonist mode, with added PTHrP, the maximal effectswere even greater (to 146% of PTHrP maximal for the C-12 compound,EU-203). This behavior demonstrates the ability of the modificationdescribed herein to vary the fundamental properties of the receptorinteractions. Modifications elsewhere in the molecule can be used tofurther modify the potency and character of the interaction of the drugcandidates.

Example 2-6: In Vivo Assay of the Compounds

Following the method of Frolik, C. A., et al. (2003) Bone 33: 372-379,20 male rats from Sino-British SIPPR/BK Lab Animal Ltd were acclimatedto standard laboratory conditions for a period of 7 days. Afteracclimation, the animals were sorted by age into groups of 5. Eachanimal in a group was treated with a single sc injection of eithervehicle or test agent.

Animals in two test groups were treated with 80 mcg/animal of huPTH1-34(Bachem) or 80 mcg/animal of EU-232. A fourth group was treated withEU-232 at 320 mcg/animal. Blood samples were collected via retro-orbitalvein at 0.5, 1, 2, 4, and 5 hrs. post-injection and blood samples werestored on ice prior to centrifugation and testing for blood PO₄ and Calevels.

In the 80 mcg groups (PTH and EU-232) there was a transient but notstatistically significant decrease in blood PO₄ levels in response toPTH or EU-232 and PO₄ levels did not further diminish after 1 hour. Inresponse to treatment with EU-232, the blood PO₄ levels decreased tostatistically significantly lower levels with time and the maximaldecrease (25-35% decrease from time 0 hr. level) was seen at the 5 hr.time point indicating a potent and prolonged duration of action forEU-232. No groups showed a statistically different blood Ca levelcompared to vehicle at any time point, thus there was no indication of apropensity for hypercalcemia following dosing.

In a similar manner, the analogs described herein (including compoundsof Table 1 in FIG. 1) are tested to evaluate their potency and durationof action in vivo.

The covalently modified peptides and/or proteins described herein areuseful for the prevention and treatment of a variety of diseases. PTHR1agonists are effective in the treatment of bone density diseases such aspostmenopausal or senile osteoporosis, hypoparathroidism, osteopenia,implant fixation, and certain metastatic tumors. Antagonistic analogsare suitable for treatment of hypercalcemia, especially as related tohyperparathyroidism or hypercalcemia of malignancy. PTH and PTHrPagonists can be used to mobilize proliferation of haematopoietic stemcells (HSC) in bone marrow in vivo or in vitro for use in bone marrowtransplant and in disease syndromes related to low blood cellconcentrations. Expansion post-transplant is an attractive applicationas well. Since many cells in the blood originate from HSCs, a wide rangeof applications is possible. Suitably labeled surfactant modifiedpeptides can be used as diagnostic probes.

Representative delivery regimens include oral, parenteral (includingsubcutaneous, intramuscular and intravenous injection), rectal, buccal(including sublingual), transdermal, inhalation ocular and intranasal.An attractive and widely used method for delivery of peptides entailssubcutaneous injection of a controlled release injectable formulation.Other administration routes for the application of the covalentlymodified peptides and/or proteins described herein are subcutaneous,intranasal and inhalation administration.

Example 2-7. Pharmaceutical Usage for Treatment of Osteoporosis

A human patient, with evidence of osteoporosis or osteopenia is treatedwith EU-204 by intranasal administration (200 μL) from a standardatomizer used in the art of a solution of the pharmaceutical agent inphysiological saline containing from 0.5 to 10 mg/mL of thepharmaceutical agent and containing standard excipients such as benzylalcohol. The treatment is repeated as necessary for the alleviation ofsymptoms such as bone pain, osteopenia, low bone density, or fractures.In a similar manner, a solution of EU-204, and selected excipients, inan evaporating solvent containing such as a hydrofluoroalkane isadministered intranasally by metered dose inhaler (MDI) as needed tostimulate bone accretion. The effect of treatment is measured by use ofstandard tests, including the Bone Mineral Density test (BMD test).

All of the compounds described in Table 1 of FIG. 1 are tested using asimilar protocol.

In a similar manner, administration of an adjusted amount bytransbuccal, intravaginal, inhalation, subcutaneous, intravenous,intraocular, or oral routes is tested to determine the level ofstimulation of PTHR1 on cells in the body, and to determine therapeuticeffects.

Example 3-1: Reagents—N-α-Fmoc, N-ε-(1-octylβ-D-glucuronide-6-yl)-L-lysine

In an oven-dried 250 mL Erlenmeyer flask is placed 1-octylβ-D-glucuronic acid (Carbosynth Ltd., 3.06 g, 10 mmol), 50 mL anhydrousDMF, and anhydrous 1-hydroxybenzotriazole (1.62 g, 12 mmol). A chilled(4° C.) solution of N, N′-dicyclohexylcarbodiimide (2.48 g, 12 mmol) in50 mL of DMF is added, with stirring, and the reaction is allowed toproceed for 5 min. The copious white precipitate of N,N′-dicyclohexylurea is filtered on a fritted glass funnel and thefiltrate is added to a solution of N-α-Fmoc-L-lysine (3.68 g, 10 mmol)in 25 ml anhydrous DMF. The reaction is allowed to proceed for 25 minwith warming to room temp or until the ninhydrin color is very faint.The reaction mixture is filtered, stripped to dryness and crystallizedfrom MeOH/Et₂O by dissolution in MeOH and slow dilution to the cloudpoint with Et₂O, followed by refrigeration. Further purification can beachieved by silica gel chromatography using a solvent gradient fromEtOAc to EtOAc/EtOH/AcOH.

In a similar manner, but substituting N-α-Boc-L-lysine is obtainedN-α-Boc,N-ε-(1-octyl β-D-glucuronide-6-yl)-L-lysine, suitable forN-terminal incorporation and cleavage to a free N-Terminus. In a similarmanner, but substituting N-α-Ac-L-lysine is obtained N-α-Ac,N-ε-(1-octylβ-D-glucuronide-6-yl)-L-lysine, suitable for incorporation at theN-terminus of a peptide with a blocked N-terminus. In a similar manner,but substituting the appropriate amount of N-α-Fmoc-L-ornithine isobtained N-α-Fmoc,N-δ-(1-octyl β-D-glucuronide-6-yl)-L-ornithine. In asimilar manner but substituting other N-mono-protected diamino acids oneobtains the corresponding reagents. Alternatively, use of a transientMe₃Si ester protecting group during the coupling and withoutpreactivation of the 1-octyl β-D-glucuronic acid provides a facile routeto the formation of the reagents. The transient Me₃Si ester is producedby reaction of the Fmoc-Lys-OH with an equimolar amount ofN,O-bis(trimethylsilyl)acetamide in dichloromethane (CH₂Cl₂). Theorganic layer contains the desired reagent as a solution in CH₂Cl₂ readyfor coupling with the 1-alkyl glucoronide as above. The filteredreaction mixture is washed with aqueous NaHSO₄ to hydrolyze the Me₃Siester, dried over MgSO₄ and solvent is removed.

Similarly, but using peracetyl or perbenzoyl 1-octyl β-D-glucuronic acidone obtains the Ac, or Bz protected form of the reagents (e.g.2,3,4-trisacetyl 1-octyl β-D-glucuronic acid, and the like, formed bytreatment with Ac₂O). Such reagents have increased stability during acidcleavage from the resin and are used when instability duringdeprotection is detected, see (Kihlberg, J., et al. (1997) MethodsEnzymol 289: 221-245) and references therein. Final deprotection of suchproducts is carried out by base-catalyzed transesterification aftercleavage, by use of MeOH/NH₃, MeOH/NaOMe, MeOH/NH₂NH₂, as describedabove.

Example 3-2: Synthetic Peptide Analogs

In general, peptide synthesis methods involve the sequential addition ofprotected amino acids to a growing peptide chain. Normally, either theamino or carboxyl group of the first amino acid and any reactive sidechain group are protected. This protected amino acid is then eitherattached to an inert solid support, or utilized in solution, and thenext amino acid in the sequence, also suitably protected, is added underconditions amenable to formation of the amide linkage. After all thedesired amino acids have been linked in the proper sequence, protectinggroups and any solid support are removed to afford the crude peptide.The peptide is desalted and purified chromatographically.

A preferred method of preparing the analogs of the physiologicallyactive truncated peptides, having fewer than about fifty amino acids,involves solid phase peptide synthesis. In this method the α-amino (Nα)functions and any reactive side chains are protected by acid- orbase-sensitive groups. The protecting group should be stable to theconditions of peptide linkage formation, while being readily removablewithout affecting the extant peptide chain. Suitable a-amino protectinggroups include, but are not limited to t-butyloxycarbonyl (Boc),benzyloxycarbonyl (Cbz), o-chlorobenzyloxycarbonyl,biphenylisopropyloxycarbonyl, t-amyloxycarbonyl (Amoc),isobornyloxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxy-carbonyl,o-nitrophenylsulfenyl, 2-cyano-t-butoxycarbonyl,9-fluorenyl-methoxycarbonyl (Fmoc) and the like, preferably Boc or morepreferably, Fmoc. Suitable side chain protecting groups include, but arenot limited to: acetyl, benzyl (Bzl), benzyloxymethyl (Bom), Boc,t-butyl, o-bromobenzyloxycarbonyl, t-butyl, t-butyldimethylsilyl,2-chlorobenzyl (Cl-z), 2,6-dichlorobenzyl, cyclohexyl, cyclopentyl,isopropyl, pivalyl, tetrahydropyran-2-yl, tosyl (Tos),2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), trimethylsilyland trityl. A preferred Nα-protecting group for synthesis of thecompounds is the Fmoc group. Preferred side chain protecting groups areO-t-Butyl group for Glu, Tyr, Thr, Asp and Ser; Boc group for Lys andTrp side chains; Pbf group for Arg; Trt group for Asn, Gln, and His. Forselective modification of a Lys residue, orthogonal protection with aprotecting group not removed by reagents that cleave the Fmoc or t-butylbased protecting groups is preferred. Preferred examples formodification of the Lys side chain include, but are not limited to,those removed by hydrazine but not piperidine; for example1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde) or1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) andallyloxycarbonyl (Alloc). The Fmoc-Lys(ivDde) or Fmoc-Lys(Dde)protecting group scheme is preferred in cases where side chain lactamformation is desired (Houston, M. E., Jr., et al. (1995) J Pept Sci 1:274-282; Murage, E. N., et al. (2010) J Med Chem), since in this caseFmoc-Glu(O-Allyl) and Fmoc-Lys(Alloc) can be incorporated and used toprovide transient protection, then deprotected for lactam formationwhile the Lys(Dde) protecting group remains for later removal andreaction with the functionalized surfactant.

The Fmoc-Lys(ivDde) or Fmoc-Lys(Dde) protecting group scheme ispreferred in cases where side chain lactam formation is desired(Houston, M. E., Jr., et al. (1995) J Pept Sci 1: 274-282; Murage, E.N., et al. (2010) J Med Chem), since in this case Fmoc-Glu(O-Allyl) andFmoc-Lys(Alloc) can be incorporated and used to provide transientprotection, then deprotected for lactam formation while the Lys(Dde)protecting group remains for later removal and reaction with thefunctionalized surfactant. The side chain lactam between acidic andbasic residue (e.g. Glu and Lys) is carried out after removal of theallyl-based protection by activation of the carboxyl side chain functionwith N,N′-diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) or2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate(HBTU)/N,N-di-isopropylethylamine (DIEA), using standard protocols wellknown to the art.

In solid phase synthesis, the C-terminal amino acid is first attached toa suitable resin support. Suitable resin supports are those materialswhich are inert to the reagents and reaction conditions of the stepwisecondensation and deprotection reactions, as well as being insoluble inthe media used. Examples of commercially available resins includestyrene/divinylbenzene resins modified with a reactive group, e.g.,chloromethylated co-poly-(styrene-divinylbenzene), hydroxymethylatedco-poly-(styrene-divinylbenzene), and the like. Benzylated,hydroxymethylated phenylacetamidomethyl (PAM) resin is preferred for thepreparation of peptide acids. When the C-terminus of the compound is anamide, a preferred resin isp-methylbenzhydrylamino-co-poly(styrene-divinyl-benzene) resin, a 2,4dimethoxybenzhydrylamino-based resin (“Rink amide”), and the like. Anespecially preferred support for the synthesis of larger peptides arecommercially available resins containing PEG sequences grafted ontoother polymeric matricies, such as the Rink Amide-PEG and PAL-PEG-PSresins (Applied Biosystems) or similar resins designed for peptide amidesynthesis using the Fmoc protocol. Thus in certain cases it is desirableto have an amide linkage to a PEG chain. It those cases it is convenientto link an N-Fmoc-amino-PEG-carboxylic acid to the amide forming resinabove (e.g. Rink amide resin and the like). The first amino acid of thechain can be coupled as an N-Fmoc-amino acid to the amino function ofthe PEG chain. Final deprotection will yield the desiredPeptide-NH-PEG-CO—NH₂ product.

Attachment to the PAM resin may be accomplished by reaction of the Nαprotected amino acid, for example the Boc-amino acid, as its ammonium,cesium, triethylammonium, 1,5-diazabicyclo-[5.4.0]undec-5-ene,tetramethylammonium, or similar salt in ethanol, acetonitrile,N,N-dimethylformamide (DMF), and the like, preferably the cesium salt inDMF, with the resin at an elevated temperature, for example betweenabout 40° and 60° C., preferably about 50° C., for from about 12 to 72hours, preferably about 48 hours. This will eventually yield the peptideacid product following acid cleavage or an amide following aminolysis.

The Nα-Boc-amino acid may be attached to the benzhydrylamine or HMP Amresin by means of, for example, an N,N′-diisopropylcarbodiimide(DIC)/1-hydroxybenzotriazole (HOBt) mediated coupling for from about 2to about 24 hours, preferably about 2 hours at a temperature of betweenabout 10° and 50° C., preferably 25° C. in a solvent such as CH₂Cl₂ orDMF, preferably CH₂Cl₂.

For Boc-based protocols, the successive coupling of protected aminoacids may be carried out by methods well known in the art, typically inan automated peptide synthesizer. Following neutralization withtriethylamine, N,N-di-isopropyl ethylamine (DIEA), N-methylmorpholine(NMM), collidine, or similar base, each protected amino acid isintroduced in approximately about 1.5 to 2.5 fold molar excess and thecoupling carried out in an inert, nonaqueous, polar solvent such asCH₂Cl₂, DMF, N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA), ormixtures thereof, preferably in dichloromethane at ambient temperature.For Fmoc-based protocols no acid is used for deprotection but a base,preferably DIEA or NMM, is usually incorporated into the couplingmixture. Couplings are typically done in DMF, NMP, DMA or mixedsolvents, preferably DMF. Representative coupling agents areN,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropyl-carbodiimide (DIC)or other carbodiimide, either alone or in the presence of HOBt, O-acylureas, benzotriazol-1-yl-oxytris(pyrrolidino)phosphoniumhexafluorophosphate (PyBop), N-hydroxysuccinimide, otherN-hydroxyimides, or oximes. Alternatively, protected amino acid activeesters (e.g. p-nitrophenyl, pentafluorophenyl and the like) orsymmetrical anhydrides may be used. Preferred coupling agents are of theaminium/uronium (alternative nomenclatures used by suppliers) class suchas 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (HBTU),O-(7-azabenzotraiazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU),2-(6-Chloro-1H-benzotraiazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (HCTU), and the like.

A preferred method of attachment to the Fmoc-PAL-PEG-PS resin may beaccomplished by deprotection of the resin linker with 20% piperidine inDMF, followed by reaction of the N-α-Fmoc protected amino acid, about a5 fold molar excess of the N-α-Fmoc-amino acid, using HBTU:di-isopropylethylamine (DIEA) (1:2) in DMF in a microwave-assistedpeptide synthesizer with a 5 min, 75° max coupling cycle.

For this Fmoc-based protocol in the microwave-assisted peptidesynthesizer, the N-α-Fmoc amino acid protecting groups are removed with20% piperidine in DMF containing 0.1M 1-hydroxybenzotriazole (HOBt), ina double deprotection protocol for 30 sec and then for 3 min with atemperature maximum set at 75° C. HOBt is added to the deprotectionsolution to reduce aspartimide formation. Coupling of the next aminoacid then employs a five-fold molar excess using HBTU:DIEA (1:2) with a5 min, 75° max. double-coupling cycle.

At the end of the solid phase synthesis the fully protected peptide isremoved from the resin.

When the linkage to the resin support is of the benzyl ester type,cleavage may be effected by means of aminolysis with an alkylamine orfluoroalkylamine for peptides with an alkylamide C-terminus, or byammonolysis with, for example, ammonia/methanol or ammonia/ethanol forpeptides with an unsubstituted amide C-terminus, at a temperaturebetween about =10° and 50° C., preferably about 25° C., for betweenabout 12 and 24 hours, preferably about 18 hours. Peptides with ahydroxy C-terminus may be cleaved by HF or other strongly acidicdeprotection regimen or by saponification. Alternatively, the peptidemay be removed from the resin by transesterification, e.g., withmethanol, followed by aminolysis or saponification. The protectedpeptide may be purified by silica gel or reverse-phase HPLC.

The side chain protecting groups may be removed from the peptide bytreating the aminolysis product with, for example, anhydrous liquidhydrogen fluoride in the presence of anisole or other carbonium ionscavenger, treatment with hydrogen fluoride/pyridine complex, treatmentwith tris(trifluoroacetyl)boron and trifluoroacetic acid, by reductionwith hydrogen and palladium on carbon or polyvinylpyrrolidone, or byreduction with sodium in liquid ammonia, preferably with liquid hydrogenfluoride and anisole at a temperature between about −10° and +10° C.,preferably at about 0° C., for between about 15 minutes and 2 hours,preferably about 1.5 hours.

For peptides on the benzhydrylamine type resins, the resin cleavage anddeprotection steps may be combined in a single step utilizing liquidhydrogen fluoride and anisole as described above or preferably throughthe use of milder cleavage cocktails. For example, for the PAL-PEG-PSresin, a preferred method is through the use of a double deprotectionprotocol in the microwave-assisted peptide synthesizer using one of themild cleavage cocktails known in the art, such asTFA/water/tri-iso-propylsilane/3,6-dioxa-1,8-octanedithiol (DODT)(92.5/2.5/2.5/2.5) for 18 min at 38° C. each time. Cleavage of alkylglycoside containing materials have shown survival of the alkylglycoside linkage using protocols with TFA/water ratios in the 9/1 to19/1 range. A typical cocktail is 94% TFA: 2% EDT; 2% H₂O; 2% TIS.Typically the fully deprotected product is precipitated and washed withcold (−70° to 4° C.) Et₂O, dissolved in deionized water and lyophilized.

The peptide solution may be desalted (e.g. with BioRad AG-3® anionexchange resin) and the peptide purified by a sequence ofchromatographic steps employing any or all of the following types: ionexchange on a weakly basic resin in the acetate form; hydrophobicadsorption chromatography on underivatizedco-poly(styrene-divinylbenzene), e.g. Amberlite® XAD; silica geladsorption chromatography; ion exchange chromatography oncarboxymethylcellulose; partition chromatography, e.g. on Sephadex®G-25; counter-current distribution; supercritical fluid chromatography;or HPLC, especially reversed-phase HPLC on octyl- oroctadecylsilylsilica (ODS) bonded phase column packing.

Also provided herein are processes for preparing covalently modifiedpeptides and/or proteins described herein and pharmaceuticallyacceptable salts thereof, which processes comprise sequentiallycondensing protected amino acids on a suitable resin support, removingthe protecting groups and resin support, and purifying the product, toafford analogs of the physiologically active truncated homologs andanalogs of the covalently modified peptides and/or proteins describedherein. In some embodiments, covalently modified peptides and/orproteins described herein incorporate alkyl glycoside modifications asdefined above.

Another aspect relates to processes for preparing covalently modifiedpeptides and/or proteins described herein and pharmaceuticallyacceptable salts thereof, which processes comprise the use ofmicrowave-assisted solid phase synthesis-based processes or standardpeptide synthesis protocols to sequentially condense protected aminoacids on a suitable resin support, removing the protecting groups andresin support, and purifying the product, to afford analogs of thephysiologically active peptides, as defined above.

Example 3-3. General Oxidation Method for Uronic Acids

To a solution of 1-dodecyl β-D-glucopyranoside (Carbosynth) [2.0 g, 5.74mmol]_in 20 mL of acetonitrile and 20 mL of DI water was added(diacetoxyiodo)benzene (Fluka) [4.4 g, 13.7 mmol] and TEMPO(SigmaAldrich) [0.180 g, 1.15 mmol]. The resulting mixture was stirredat room temperature for 20 h. The reaction was followed by massspectrometry (for example LCQ ESI) and upon completion, the reactionmixture was diluted with water and lyophilized to dryness to give 1.52 g(crude yield 73.1%) of the crude product, 1-dodecyl β-D-glucuronic acid,as a white powder, which was used directly for the solid phase synthesiswithout further purification. This product was previously prepared by analternative process using NaOCl as oxidant, as described in thespecification, and also has been used for longer alkyl groups. In asimilar manner are prepared the desired alkyl saccharide uronic acidsused to make the products and reagents described herein.

In a like manner, but using the corresponding 1-tetradecyl, 1-hexadecyl,and 1-octadecyl 3-D-glucopyranosides (purchased from Anatrace, Maumee,Ohio) were prepared the desired 1-alkyl saccharide uronic acids whichwere used to make the products and reagents described herein. For longeralkyl groups 1,4-dioxane was used instead of acetonitrile and thetemperature was raised as high as 30° C.

In a like manner, but using, for example, the corresponding 1-octyl,1-decyl, 1-undecyl, 1-tetradecyl, 1-hexadecyl, and 1-octadecylglycosides (purchased from Anatrace, Maumee, Ohio) were prepared thedesired 1-alkyl saccharide uronic acids which were used to make theproducts and reagents described herein. In a like manner, but using, forexample, the corresponding 1-octyl, 1-decyl, 1-undecyl, 1-tetradecyl,1-hexadecyl, and 1-octadecyl β-D-melibiosides or β-D-maltosides(purchased from Anatrace, Maumee, Ohio) were prepared the desired1-alkyl disaccharide uronic acids which were used to make the productsand reagents described herein. In each case the corresponding alphaglycosides can be treated similarly

Example 3-4: Preparation of Analog EU-A387

A sample ofFmoc-His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Bip-Ser-Lys-Tyr-Leu-Glu-Ser-Lys(Alloc)-Rinkamide resin was prepared by sequential addition of N-alpha-Fmocprotected amino acids as described in Example 1 and deprotected on theLys-N-epsilon position by incubation with Pd(PPh₃)₄ (0.5 eq) and DMBA(20 eq) in DMF/CH₂Cl₂ (1:1) overnight in the dark at room temperature.Following washing by DMF/CH₂Cl₂, the Lys side chain was acylated with1′-dodecyl β-D-glucuronic acid in DMF/CH₂Cl₂ through the use ofDIC/HOBt. Completion of the coupling was checked by ninhydrin and theproduct was washed extensively with CH₂Cl₂.

The product resin was submitted to final deprotection and cleavage fromthe resin by treatment with the cleavage cocktail (94% TFA: 2% EDT; 2%H₂O; 2% TIS) for a period of 240 min at room temperature. The mixturewas treated with Et₂O, to precipitate the product and washed extensivelywith Et₂O to yield the crude title peptide product after drying invacuo.

Purification was carried out in two batches by reversed phase (C18)hplc. The crude peptide was loaded on a 4.1×25 cm hplc column at a flowrate of 15 mL/min (15% organic modifier; acetic acid buffer) and elutedwith a gradient from 15-45% buffer B in 60 min at 50° C. The productfraction was lyophilized to yield the title product peptide with apurity 98.03% by analytical hplc (18.6 min; 30-60% CH₃CN in 0.1%TFA)/mass spectrometry (M+1 peak=2382.14).

Example 3-5: Preparation of Analog EU-A1024

A sample ofBoc-His(Trt)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-Leu-Asp(OtBu)-Glu(O-Allyl)-Gln(Trt)-Ala-Ala-Lys(Alloc)-Glu(O-tBu)-Phe-Ile-Lys(Dde)-Trp(Boc)-Leu-Leu-Gln(Trt)-Thr(tBu)-HMPamide resin (started from Fmoc-Thr(tBu)-HMP Am Resin, substitution 0.45mmol/g) was prepared by sequential addition of N-alpha-Fmoc protectedamino acids as described in Example 1. The allyl based side chains onGlu and Lys were deprotected by incubation with Pd(PPh₃)₄ (0.5 eq) andDMBA (20 eq) in DMF/CH₂Cl₂ (1:1) overnight in the dark at roomtemperature. The resin was washed with 0.5% DIEA in DMF (twice), 0.5%sodium diethyldithiocarbamate in DMF (twice) and DMF/CH₂Cl₂ until alight yellow resin was obtained. The side chain lactam linkage wasformed by coupling the Glu and Lys with DIC/HOBT (5 equivalents) in DMF.The reaction was checked for completeness with ninhydrin and recoupledif necessary. Following washing by DMF/CH₂Cl₂, the Lys side chain wasdeprotected by incubation with 5% hydrazine hydrate in DMF (10equivalents) twice, in each case for 15 min. Following washing byDMF/CH₂Cl₂ the side chain amino group of the deprotected Lys residue wasreacted with 1′-tetradecyl β-D-melibiouronic acid in DMF/CH₂Cl₂ throughthe use of DIC/HOBt. Completion of the coupling was checked by ninhydrinand the product was washed extensively with CH₂Cl₂. Any couplings whichwere not complete by ninhydrin were rerun. In general, 10-12 g ofpeptide product resin was obtained from a 2 mmole synthesis.

The product resin is submitted to final deprotection and cleavage fromthe resin by treatment with the cleavage cocktail (94% TFA: 2% EDT; 2%H₂O; 2% TIS) for a period of 240 min at room temperature. The mixturewas treated with Et₂O, to precipitate the product and washed extensivelywith Et₂O to yield the crude title peptide product after drying invacuo. In general, 5 to 8 g crude product peptide was obtained.

Purification was carried out in two batches by reversed phase (C18)hplc. The crude peptide (1-1.5 g) was loaded on a 4.1×25 cm hplc columnat a flow rate of 15 mL/min (15% organic modifier; 0.1% TFA buffer) andeluted with a gradient from 35-55% buffer B in 70 min at roomtemperature. Repurification of the less pure fractions was done for thefractions with a purity of >70%. The product fraction was lyophilized toyield the title product peptide with a purity 98.7% by analytical hplc(10.3 min; 45-75% CH₃CN in 0.1% TFA)/mass spectrometry (1317.67, +3charged; 1976.13, +2 charged; molecular weight 3950.44). In a similarmanner were prepared the other analogs of the invention, thecharacterization of which is illustrated below.

The corresponding 1-methyl and 1-octyl analogs of the title compound areprepared in a similar manner, but using the reagents 1′-methylβ-D-glucuronic acid and 1′-octyl β-D-glucuronic acid (Carbosynth). In alike manner, but using the corresponding 1-octyl, 1-decyl, 1-undecyl,1-tetradecyl, 1-hexadecyl, and 1-octadecyl 13-D-glucouronic acids(prepared as described above) were preprared the products of theinvention. In a like manner, but using the corresponding 1-octyl,1-decyl, 1-undecyl, 1-tetradecyl, 1-hexadecyl, and 1-octadecylβ-D-melibiouronic or β-D-maltouronic (prepared as described above) wereprepared the products and reagents described herein. Alternatively, the1-alkyl glucuronyl, or other uronic acylated analogs, may be prepared byinitial purification of the deprotected or partially deprotected peptidefollowed by acylation by the desired uronic acid reagent.

Analysis and characterization was done by HPLC/mass spectrometry inpositive ion mode using the eluent gradients given in the table below.Analysis and characterization was done by HPLC/mass spectrometry inpositive ion mode using the eluent gradients given in the table below.

Compound Molecular Wt Molecular Wt HPLC Name Expected Found (min;elution) EU-A387 2379.66 2380.14 18.6 [b] EU-A388 2393.69 2393.74 16.0[a] EU-A391 2317.62 2318.26 11.2 [b] EU-A455 2988.36 2988.00 11.5 [b]EU-A474 2570.86 2570.54 11.3 [b] EU-A478 2459.75 2459.74 11.1 [b]EU-A484 2544.86 2545.06  9.6 [b] EU-A501 2904.2 2903.34  7.9 [b] EU-A5022776.07 2776.14  8.0 [b] EU-A503 2704.98 2704.40  8.0 [b] EU-A5042548.80 2548.00  9.1 [b] EU-A505 2392.61 2392.40 10.5 [b] EU-A5062305.53 2305.06 10.7 [b] EU-A507 3763.23 3762.66  9.0 [b] EU-A5212303.56 2303.60  8.2 [c] EU-A522 2315.60 2315.60 14.2 [d] EU-A5232615.94 2616.00  8.1 [b] EU-A524 2459.75 2459.74 12.7 [d] EU-A5252459.75 2459.06  6.0 [c] EU-A526 2473.75 2473.60 12.7 [d] EU-A5272390.64 2390.40 14.6 [d] EU-A529 2546.83 2546.80  9.5 [b] EU-A5312546.83 2546.80  9.5 [b] EU-A532 2559.00 2558.66  9.6 [b] EU-A5332560.96 2560.66  9.5 [b] EU-A534 2544.99 2544.94  9.7 [b] EU-A5352573.05 2574.00 12.0 [b] EU-A536 2602.96 2603.46 14.3 [b] EU-A5382516.99 2516.40 10.3 [b] EU-A539 2657.20 2656.80 10.8 [b] EU-A5402685.20 2684.94  9.8 [c] EU-A541 2713.20 2712.80 13.0 [c] EU-A5442631.94 2632.26 10.8 [b] EU-A546 2687.94 2688.80  9.1 [c] EU-A5492388.67 2388.66  6.3 [e] EU-A551 2444.67 2445.20 11.4 [e] EU-A5522472.67 2473.14 10.7 [f] EU-A554 2560.86 2560.40 10.3 [c] EU-A5562616.86 2616.40 11.7 [e] EU-A557 2644.86 2645.74 10.4 [f] EU-A5602570.86 2571.06  8.3 [c] EU-A562 2626.86 2626.66  9.9 [e] EU-A5632654.86 2655.06  8.7 [f] EU-A565 2542.80 2542.54  9.5 [c] EU-A5672598.80 2599.06 12.0 [e] EU-A568 2626.80 2626.54 10.1 [f] HPLC gradientsin 0.1% TFA [a] 35 to 65% CH₃CN over 30 min. [b] 30 to 60% CH₃CN over 20min. [c] 35 to 65% CH₃CN over 20 min. [d] 25 to 55% CH₃CN over 20 min.[e] 40 to 70% CH₃CN over 20 min. [f] 45 to 75% CH₃CN over 20 min. HPLCon Phenomenex Luna C18 5 micron 250 × 4.6 mm.Additional compounds synthesized and analyzed as described above are:

Compound Molecular Wt Molecular Wt HPLC Name Expected Found (min;elution) EU-A570 2656.16 2656.00 10.4 [b] EU-A571 2684.16 2683.34 11.2[c] EU-A575 2670.16 2670.94 11.8 [b] EU-A576 2698.16 2697.20 11.2 [c]EU-A580 2668.20 2667.20 12.3 [b] EU-A581 2696.20 2695.46 11.1 [c]EU-A592 2724.20 2724.58  9.9 [e] EU-A595 2682.20 2682.40  9.7 [c]EU-A596 2710.20 2710.46 10.4 [c] EU-A597 2738.20 2738.18 10.9 [e]EU-A721 2461.85 2461.74 10.3 [b] EU-A722 2475.85 2475.34 10.8 [b]EU-A723 2459.88 2459.86  7.7 [c] EU-A724 2473.88 2473.34 11.1 [b]EU-A725 2471.92 2472.00 10.8 [b] EU-A726 2557.03 2556.80 11.0 [b]EU-A727 2485.92 2485.74 10.9 [b] EU-A728 2513.92 2513.86 10.6 [c]EU-A729 2541.92 2541.86  9.7 [e] EU-A730 2569.92 2569.74  9.4 [f]EU-A731 2425.88 2425.32 10.6 [d] EU-A732 2476.95 2476.40  9.4 [c]EU-A733 2381.83 2382.02 11.4 [b] EU-A734 2616.09 2616.18 11.4 [b]EU-A750 1611.89 1611.56  9.4 [c] EU-A751 1625.89 1625.35  9.7 [c]EU-A752 1709.93 1709.41 11.7 [g] EU-A753 1637.84 1637.46 12.5 [b]EU-A754 1651.84 1651.18  9.9 [c] EU-A755 1711.93 1711.46 10.3 [h]EU-A756 1671.98 1671.37  9.8 [e] EU-A757 1770.02 1769.17 14.9 [g]EU-A770 3333.61 3334.65  9.5 [b] EU-A771 3678.25 3677.96 11.3 [c]EU-A772 3762.25 3763.35 14.9 [e] EU-A773 3790.31 3791.31 11.1 [f]EU-A774 3475.74 3477.15 10.5 [d] EU-A775 3820.38 3821.52 10.4 [c]EU-A776 3904.38 3905.76 10.2 [f] EU-A777 3932.44 3933.69  9.7 [f]EU-A792 3793.43 3793.52  8.9 [f] EU-A793 3821.43 3821.60 10.8 [f]EU-A794 3849.43 3848.78 10.5 [g] EU-A945 3777.18 3777.54 12.2 [e]EU-A948 3861.18 3862.14 13.0 [f] EU-A993 3759.22 3759.00  9.3 [f]EU-A994 3787.22 3787.44 11.4 [f] EU-A995 3815.22 3815.52 14.5 [g]EU-A996 3843.22 3843.12 12.3 [g] EU-A999 3935.35 3934.66 13.4 [e]EU-A1011 3854.21 3854.38 14.2 [c] EU-A1017 3896.26 3895.77 12.3 [c]EU-A1023 3921.35 3921.15 10.5 [e] EU-A1024 3950.44 3949.95 10.3 [f]EU-A1026 4005.55 4004.64 12.8 [g] EU-A1029 3939.31 3939.06 10.7 [e]EU-A1032 4023.51 4025.22 13.9 [f] EU-A1035 3840.21 3838.21  9.8 [e]EU-A1041 3882.26 3880.50  9.6 [e] EU-A1044 3966.46 3965.46 12.6 [f]EU-A1167 3731.15 3731.42  8.9 [f] EU-A1168 3745.15 3745.20 10.8 [i]EU-A1173 3978.49 3977.00 12.9 [f] HPLC gradients in 0.1% TFA [a] 35 to65% CH₃CN over 30 min. [b] 30 to 60% CH₃CN over 20 min. [c] 35 to 65%CH₃CN over 20 min. [d] 25 to 55% CH₃CN over 20 min. [e] 40 to 70% CH₃CNover 20 min. [f] 45 to 75% CH₃CN over 20 min. [g] 50 to 80% CH₃CN over20 min. [h] 10 to 40% CH₃CN over 20 min. [i] 30 to 90% CH₃CN over 20min. HPLC on was carried out on a Phenomenex Luna C18 5 micron 250 × 4.6mm analytical column.

Example 3-6: Cellular Assay of the Compounds

Compounds were weighed precisely in an amount of approximately 1 mg andassayed in standard cellular assays (Cerep SA). The readout is theamount of cAMP generated in the cells treated with the test compounds,in either agonist or antagonist mode. The assay used was the stimulationof cAMP levels in the glucagon (human, cloned into CHO cells) and GLP-1(murine cell line) cellular assays. The assays are described in Chicchi,G. G., et al. (1997) J Biol Chem 272: 7765-7769 and Runge, S., et al.(2003) Br J Pharmacol 138: 787-794.

For compound EU-A391 the GLCR cellular response does not change and theGLP1R cellular response rises steeply with and EC50 of 420 nM

EC₅₀ EC₅₀ murine GLP-1 R glucagon R Compound Structure (nM) (nM) EU-A3911-dodecyl 420 n.c. EU-A455 1-dodecyl 59 770 EU-A474 1-dodecyl 3000 n.c.EU-A478 1-dodecyl n.c. n.c. EU-A484 1-dodecyl n.c. n.c. EU-A5011-dodecyl 20000 12000 EU-A502 1-dodecyl 9400 n.c. EU-A503 1-dodecyl n.c.n.c. EU-A504 1-dodecyl 3100 1100 EU-A505 1-dodecyl 8500 6100 EU-A5061-dodecyl 4600 1300 EU-A507 1-dodecyl 18 1 EU-A521 1-dodecyl n.c. n.c.EU-A522 1-dodecyl n.c. 9000 EU-A523 1-dodecyl n.c. n.c. EU-A5241-dodecyl n.c. n.c. EU-A525 1-dodecyl n.c. n.c. EU-A526 1-dodecyl n.c.n.c. EU-A527 1-dodecyl n.c. 5000 EU-A529 1-dodecyl n.c. 7000 EU-A5311-dodecyl 2100 1100 EU-A532 1-dodecyl 5000 2600 EU-A533 1-dodecyl 770780 EU-A534 1-dodecyl 290 1900 EU-A535 1-tetradecyl §4800 2100 EU-A5361-hexadecyl >10000 4400 EU-A538 1-dodecyl 270 n.c. EU-A539 1-dodecyl 8602300 EU-A540 1-tetradecyl n.c. 8800 EU-A541 1-hexadecyl 800 5000 n.c.means EC50 not calculable §means superagonistA further series of cellular assays were carried out using standardcellular assays (DiscoveRx, LeadHunter assays) using readout of cAMPstimulation or arrestin activation. Compounds were weighed precisely inan amount of approximately 1 mg and shipped to DiscoveRx for dilutionand assay. The assay used were for the glucagon (human, cloned into CHOcells) and GLP-1 (human, cloned into CHO cells) receptors in cellularassays.

EC₅₀ cAMP EC₅₀ arrestin EC₅₀ cAMP EC₅₀ arrestin GLP-1 R GLP-1 R glucagonR glucagon R Compound (nM) (nM) (nM) (nM) EU-A507 0.01 9 0.02 100EU-A534 87 1100 EU-A538 55 3500 EU-A750 >1000 >1000 EU-A751 >1000 >1000EU-A752 146 >1000 EU-A753 >1000 >1000 EU-A754 360 >1000 EU-A755 486 471EU-A756 611 >1000 EU-A757 6.7 >1000 EU-A770 0.01 2.3 0.5 >100 EU-A7710.07 14.2 0.4 >100 EU-A772 0.07 8.4 5.4 >100 EU-A773 0.08 8.3 1.5 >100EU-A774 0.009 6.8 0.15 22.7 EU-A775 0.16 17 0.3 33.6 EU-A776 1.2 >1006.5 >100 EU-A777 0.1 34.5 0.6 73 EU-A792 <0.05 27.9 <0.05 EU-A793 <0.0523.8 <0.05 EU-A794 0.05 59.4 0.18 EU-A945 <0.05 9.4 0.06 EU-A948 0.0825.6 9.1 EU-A992 0.009 0.019 EU-A993 <0.05 12.3 <0.05 EU-A994 0.05 10.2<0.05 EU-A995 0.035 59.5 0.15 EU-A996 0.05 >100 0.87 EU-A999 0.05 0.015EU-A1011 0.16 0.51 EU-A1017 0.44 0.10 EU-A1023 0.028 0.035 EU-A10290.019 0.06 EU-A1032 0.03 5.4 EU-A1035 0.02 0.19 EU-A1041 0.02 0.13EU-A1044 0.07 0.57 EU-A1167 0.013 0.019 EU-A1168 0.07 0.14

Example 3-7: In Vivo Assay of Compounds—Db/Db Mice

The 60 female db/db B6BKS (D) Leprdb/J (strain 000697) mice for thisstudy were approximately 8 to 9 weeks old at arrival (JacksonLaboratory, Bar Harbor, Me.). The mice were randomized by weight and twotreatment groups of 8 female mice each were administered the testarticles, EU-A994, EU-A995, or EU-A1026, at dose levels of 100 or 300nmoles/kg. One group of 8 female mice served as the vehicle control andreceived the vehicle, 0.2% BSA in saline, pH 7.4. One additional groupof 8 female mice received the positive control article, liraglutide, ata dose level of 50 nmoles/kg. The test articles, vehicle, and positivecontrol article were administered on Day 1, at approximately 0, 7, and24 hours during the study via subcutaneous injection at a dose volume of6 mL/kg.

Clinical observations were conducted at receipt, prior to randomization,and daily from Days 1 to 5. Body weights were measured and recorded atreceipt, prior to randomization, and daily from Days 1 to 5. Foodconsumption was measured and recorded daily from Days 1 to 5. Bloodsamples for glucose analysis were collected pretest (Day −3) and at 0,1, 2, 4, 8, 10, 24, 48, 72, and 96 hours following the first dose onDay 1. At study termination all animals were euthanized and thecarcasses were discarded without further evaluation.

Significant body weight changes were noted against vehicle forliraglutide and high dose EU-A994 and high dose EU-A1026 on Days 2 and 3and low dose EU-A1026 on Days 3 and 4. In the food consumption analysis,liraglutide-treated animals were significantly different from vehicle onDays 1 and 2, high dose EU-A994 at Day 1, low dose EU-A995 at Day 1 and2, high dose EU-A994 at Day 1, and low and high dose of EU-A1026 at Day2 were significantly different from Liraglutide. Glucose levels forLiraglutide at 10 hours and high dose EU-A994 at 10 and 24 hours weresignificantly different from vehicle. Low dose EU-A995 and EU-A1026 at10 hours were significantly different from liraglutide (FIG. 5). In alike manner, other analogs from the series were tested for effects onblood glucose, body weight and food consumption.

db/db mice - Mean Dose Blood Glucose (mg/dL) Compound (nmole/kg) 0 hr 10hr 24 hr 48 hr liraglutide 50 595 265 347 377 EU-A994 300 554 242 209385 EU-A995 300 503 471 493 527 EU-A1026 300 531 399 438 505 liraglutide50 528 286 404 477 EU-A993 250 503 305 324 426 EU-A995 250 539 584 548546 EU-A1023 50 507 310 355 375 EU-A1023 250 508 264 194 375 liraglutide50 442 174 244 439 EU-A992 250 405 104 112 130 EU-A1167 250 379 171 117113 EU-A1168 250 380 133 169 164

Example 3-8: In Vivo Assay of Compounds

Sixty (60) diet induced obese C57BL/6J male mice are received from JAXlabs at 14 wks of age. The mice are ear notched for identification andhoused in individually and positively ventilated polycarbonate cageswith HEPA filtered air at density of one mouse per cage. The animal roomis lighted entirely with artificial fluorescent lighting, with acontrolled 12 h light/dark cycle. The normal temperature and relativehumidity ranges in the animal rooms are 22+4° C. and 50+15%,respectively. Filtered tap water, acidified to a pH of 2.8 to 3.1, andhigh fat diet (60 kcal %) are provided ad libitum.

Following a 2 week acclimation, 40 mice are chosen based on desired bodyweight range and mice are randomized into groups (n=10) as below.Group 1. Vehicle treated; Group 2. Low dose test cmpd; Group 3. Mid dosetest cmpd; Group 4. High dose test cmpd. Mice are dosed via SC daily for28 days. Body weights and cage side observations are recorded daily.Food and water intake will be recorded weekly. Mice undergo NMRmeasurements for determining whole body fat and lean composition on days1 (pre dose) and 26. On days 0, 14 and 27, mice are fasted overnight foran oral glucose tolerance test. Next day, the first blood sample iscollected via tail nick (t=0). Mice are then administered a bolus of 1.0g/kg glucose. Blood samples are obtained via tail nick at 5, 30, 60 and120 min after glucose and plasma glucose will be immediately determinedusing a glucometer.

Sacrifice and tissue collection: Mice are sacrificed on day 29. Terminalblood is processed to serum/plasma and aliquots are sent for analysis ofglucose, insulin and lipid profile. Body composition is determined byNMR. The optimal compound profile shows decreased glucose excursion inthe OGTT, decreased basal insulin secretion, with potentiatedglucose-dependent insulin secretion, decreased weight gain, decreasedfat mass but minimal effects on lean mass.

Example 3-9: Plasma Protease Stability

Bioanalytical Method Development Summary I. Instruments used, API-4000Mass Spectrometer, ESI positive, MRM Scan; Shimadzu HPLC/CTC Autosamplerwith ACE C8 column (2.1×50 mm, 5 μm), Mobile Phase A: 0.1% Formic acid,5 mM NH₄OAc in water, Mobile Phase B: 0.1% Formic acid in CH₃CN, 10 μLof sample was injected, II. Standard and QC Sample Preparation: i. 1mg/mL stock solution was prepared in DMSO/CH₃CN (1/1), ii. Make standardworking solutions in 50% CH₃CN with the stock solution. Theconcentrations of the working solutions were 20, 50, 100, 200, 500,1000, 2000, 5000, 10000 and 20000 ng/mL, iii. Spiked 10 μL of theworking solutions into 90 μL blank plasma and vortex them, iv. Add 300μL of internal standard solution (Verapamil, 20 ng/mL in 100% CH₃CN),vortex and centrifuge, v. Transfer the supernatant into an HPLCinjection plate for loading on the HPLC column, vi. The standard sampleswere at 2, 5, 10, 20, 50, 100, 200, 500, 1000 and 2000 ng/mL. The QCsamples were at 5 (LQC), 50 (MQC), and 500 (HQC) ng/mL. For the plasmastability study, samples of EU-A993, EU-A1023 and human GLP-1 (7-36,Bachem) were prepared in human plasma (approximately 6-20 ng/mL orsimilar concentrations above the limit of quantitation) and sampled attime points t=0, 0.5, 1, 2, 4, 8 hrs during incubation at 37 deg C.Samples were treated with internal standard solution (100% CH₃CN) asdescribed above in order to precipitate the proteins and supernatentantswere loaded into an injection plate to load them onto the hplc columnfor quantitation by mass spectrometry. A plot of compound signal(amount) versus time shows rapid decrease in GlP-1 (7-36) andessentially no change over 8 hrs in the amount of intact compound in thehuman plasma for the compounds of the invention (see FIG. 15).

Example 3-10: Uses of the Compounds

The covalently modified peptides and/or proteins described herein areuseful for the prevention and treatment of a variety of diseases relatedto obesity, the metabolic syndrome, cardiovascular disease and diabetes.Suitably labeled surfactant modified peptides can be used as diagnosticprobes.

Representative delivery regimens include oral, parenteral (includingsubcutaneous, intramuscular and intravenous injection), rectal, buccal(including sublingual), transdermal, inhalation ocular and intranasal.An attractive and widely used method for delivery of peptides entailssubcutaneous injection of a controlled release injectable formulation.Other administration routes for the application of the covalentlymodified peptides and/or proteins described herein are subcutaneous,intranasal and inhalation administration.

Example 3-11. Pharmaceutical Usage for Treatment of Insulin Resistance

A human patient, with evidence of insulin or metabolic syndrome istreated with EU-A596 by intranasal administration (200 μL) from astandard atomizer used in the art of a solution of the pharmaceuticalagent in physiological saline containing from 0.5 to 10 mg/mL of thepharmaceutical agent and containing standard excipients such as benzylalcohol. The treatment is repeated as necessary for the alleviation ofsymptoms such as obesity, elevated blood glucose and the like. In asimilar manner, a solution of EU-A596, and selected excipients, in anevaporating solvent containing such as a hydrofluoroalkane isadministered intranasally by metered dose inhaler (MDI) as needed toreduce insulin resistance. The effect of treatment is determined usingstandard tests including measurement of blood glucose levels, Body MassIndex, and/or body weight and/or measurement of waist to hip ratios.

In a similar manner, administration of an adjusted amount bytransbuccal, intravaginal, inhalation, subcutaneous, intravenous,intraocular, or oral routes is tested to determine level of stimulationof GLP1R and/or GLCR on cells in the body and to determine therapeuticeffects.

SEQUENCES

Table 1 in FIG. 1 depicts compounds that were prepared by methodsdescribed herein. The specification provides sequences for SEQ. ID. Nos.1 and 149-169. Additionally, Table 1 of FIG. 1 provides SEQ. ID Numbersfor compounds EU-A101 to EU-A199 and EU-A600 to EU-A649 having SEQ. ID.NOs. 2-148, and SEQ. ID. NO. 645 respectively, as shown in Table 1 ofFIG. 1. Compounds in Table 1 of FIG. 1, and their respective SEQ. ID.NOs. shown in Table 1 of FIG. 1 are hereby incorporated into thespecification as filed.

Table 2 in FIG. 2 depicts compounds that were prepared by methodsdescribed herein. The specification provides sequences for SEQ. ID. Nos.170-174 and SEQ. ID. NOs. 283-302. Additionally, Table 2 of FIG. 2provides SEQ. ID. Numbers for compounds EU-201 to EU-299 and EU-900 toEU-908 having SEQ. ID. NOs. 175-282 respectively, as shown in Table 2 ofFIG. 2. Compounds in Table 2 of FIG. 2, and their respective SEQ. ID.NOs. shown in Table 2 of FIG. 2 are hereby incorporated into thespecification as filed.

Table 3 in FIG. 8 depicts compounds that were prepared by methodsdescribed herein. The specification provides sequences for SEQ. ID. Nos.303-305 and SEQ. ID. Nos. 619-644. Additionally, Table 3 of FIG. 8provides SEQ. ID Numbers for compounds EU-A300 to EU-A425 having SEQ.ID. NOs. 306-431 respectively, as shown in Table 3 of FIG. 8. Compoundsin Table 3 of FIG. 8, and their respective SEQ. ID. NOs. shown in Table3 of FIG. 8 are hereby incorporated into the specification as filed.

Table 4 in FIG. 9 depicts compounds that were prepared by methodsdescribed herein. The specification provides SEQ. ID. Nos. 303-305 andSEQ. ID. Nos. 619-644. Additionally, Table 4 of FIG. 9 provides SEQ. IDNumbers for compounds EU-A426 to EU-A599 having SEQ. ID. NOs. 432-618respectively, as shown in Table 4 of FIG. 9. Additionally, Table 4 ofFIG. 9 provides SEQ. ID Numbers for compounds EU-A700 to EU-A1174 havingSEQ. ID. NOs 646-1120. Compounds in Table 4 of FIG. 9, and theirrespective SEQ. ID. NOs. shown in Table 4 of FIG. 9 are herebyincorporated into the specification as filed.

1-47. (canceled)
 48. A peptide product comprising a surfactant Xcovalently attached to a peptide, the peptide comprising a linker aminoacid U and at least one other amino acid:

wherein the surfactant X is

wherein: A is a hydrophobic group which is a substituted orunsubstituted C₁-C₃₀ alkyl group, a substituted or unsubstitutedalkoxyaryl group, a substituted or unsubstituted aralkyl group, or asteroid nucleus-containing moiety; and B is a hydrophilic group which isa saccharide and is covalently attached to the peptide via the linkeramino acid U; and wherein the peptide product displays arrestin-biasedagonism or antagonism compared to the unmodified peptide not covalentlyattached to the surfactant X.
 49. The peptide product of claim 48,wherein the saccharide is a monosaccharide or disaccharide.
 50. Thepeptide product of claim 49, wherein the saccharide is glucose,galactose, mannose, melibiose, maltose, sucrose, glucuronic acid,galacturonic acid, mannouronic acid, diglucuronic acid, melibiouronicacid or maltouronic acid.
 51. The peptide product of claim 48, whereinthe hydrophobic group is a substituted or unsubstituted C₈-C₂₀ alkylgroup.
 52. The peptide product of claim 48, wherein the surfactant X isa 1-alkyl glycoside.
 53. The peptide product of claim 52, wherein the1-alkyl glycoside is 1-eicosyl beta-D-glucuronic acid, 1-octadecylbeta-D-glucuronic acid, 1-hexadecyl beta-D-glucuronic acid, 1-tetradecylbeta-D-glucuronic acid, 1-dodecyl beta-D-glucuronic acid, 1-decylbeta-D-glucuronic acid, 1-octyl beta-D-glucuronic acid, 1-eicosylbeta-D-diglucuronic acid, 1-octadecyl beta-D-diglucuronic acid,1-hexadecyl beta-D-diglucuronic acid, 1-tetradecyl beta-D-diglucuronicacid, 1-dodecyl beta-D-diglucuronic acid, 1-decyl beta-D-diglucuronicacid, 1-octyl beta-D-diglucuronic acid, 1-eicosyl beta-D-melibiouronicacid, 1-octadecyl beta-D-melibiouronic acid, 1-hexadecylbeta-D-melibiouronic acid, 1-tetradecyl beta-D-melibiouronic acid,1-dodecyl beta-D-melibiouronic acid, 1-decyl beta-D-melibiouronic acidor 1-octyl beta-D-melibiouronic acid, or functionalized 1-eicosylbeta-D-glucose, 1-octadecyl beta-D-glucose, 1-hexadecyl beta-D-glucose,1-tetradecyl beta-D-glucose, 1-dodecyl beta-D-glucose, 1-decylbeta-D-glucose, 1-octyl beta-D-glucose, 1-eicosyl beta-D-maltoside,1-octadecyl beta-D-maltoside, 1-hexadecyl beta-D-maltoside, 1-tetradecylbeta-D-maltoside, 1-dodecyl beta-D-maltoside, 1-decyl beta-D-maltoside,1-octyl beta-D-maltoside, 1-eicosyl beta-D-melibiose, 1-octadecylbeta-D-melibiose, 1-hexadecyl beta-D-melibiose, 1-tetradecylbeta-D-melibiose, 1-dodecyl beta-D-melibiose, 1-decyl beta-D-melibioseor 1-octyl beta-D-melibiose, or the corresponding alpha-anomer.
 54. Thepeptide product of claim 48, wherein the peptide is an opioid peptide, aPTH peptide, a PTHrP peptide, a glucagon peptide or a GLP-1 peptide,each having a native or non-native amino acid sequence or structure. 55.The peptide product of claim 48, wherein the saccharide group of thesurfactant is attached to the peptide via an amide bond.
 56. Apharmaceutical composition comprising a therapeutically effective amountof a peptide product of claim 48 or a pharmaceutically acceptable saltthereof, and at least one pharmaceutically acceptable carrier orexcipient.
 57. A method of modulating signal transduction mediated by anarrestin, comprising contacting a cell with a peptide product of claim48.
 58. A method of modulating signal transduction mediated by a Gprotein-coupled receptor (GPCR), comprising contacting a cell with apeptide product of claim 48.